Method for treating breast cancer

ABSTRACT

Breast cancer cells lacking ER protein expression, PgR protein expression and/or showing absence of HER2 protein over-expression (i.e., triple-negative breast cancer cells, basal-like) can be efficiently targeted with an anti-KAAG1 antibody and killed upon delivery of a therapeutic moiety. Antibodies and antigen binding fragments that specifically binds to KAAG1 may thus be used for the, detection and therapeutic treatment of breast cancer cells that are negative for at least one of these markers. The use of antibody conjugates in the treatment of triple-negative breast cancer and/or basal-like breast cancer is disclosed herein.

BACKGROUND

World wide, greater than 1 million women are diagnosed with breastcancer each year. Breast cancer is a very heterogeneous disease made upof dozens of different types that are distinguished using a histologicalclassification system. A large subtype and a majority of cases arehistologically identified as luminal A or luminal B which can be grosslycharacterized as exhibiting estrogen receptor (ER) expression with lowgrade or higher grade histology, respectively (Santana-Davila and Perez,2010). Immunohistochemical methods are used to measure the expression ofprogesterone receptor (PgR) which, when coupled with ER-positive statusallows the classification of a tumor as being hormone responsive.Furthermore, the over-expression or amplification of human epidermalgrowth factor receptor 2 (HER2) can be monitored either withimmunohistochemistry or fluorescence in situ hybridization (FISH).Generally, the expression of these three markers in breast tumors isassociated with a better clinical outcome because there are severaltreatment options available for patients that target these proteins (deRuijter et al., 2011), including tamoxifen, Arimidex™ (anastrozole),Aromasin™ (exemestane), Femara™ (letrozole), Faslodex™ (fulvestrant),Herceptin™ (trastuzumab) or Tykerb™ (lapatinib).

Another histological subtype of breast cancer consists of the basal-likecancers which are associated with, among others, a higher histologicalgrade, increase mitotic index and high Ki67 expression (Santana-Davilaand Perez, 2010). The vast majority of basal-like cancers are comprisedof triple-negative breast cancer (TNBC) cases, which make up a between15-20% of all diagnosed breast cancer cases (Ismail-Khan and Bui, 2010).TNBC is defined by the lack of protein expression of ER, PgR and theabsence of HER2 protein over-expression. The relationship betweenbasal-like cancer and TNBC is not easily delineated since not all TNBCare basal-like and not all basal-like cancers are TNBC, butapproximately 75% of cases in these categories share characteristics ofboth. TNBC is associated with poor prognosis consisting of low five-yearsurvival rates and high recurrence.

Patients with TNBC develop their disease earlier in life compared withother breast cancer subtypes and are often diagnoses at thepre-menopausal stage (Carey et al., 2006). Triple-negative breast cancershows an increased propensity of recurrence after treatment and seem tobe more aggressive than other breast carcinoma subtypes (Nofech-Mozes etal., 2009), similar to those of the basal-like breast cancer subtype.Consequently, the overall five-year survival of TNBC patients issignificantly lower than those diagnosed with other subtypes of breastcancer. There is currently no acceptable specific molecular marker forTNBC. Despite this lack, these tumors do respond to chemotherapy (Kriegeet al., 2009). Patients have shown better response to cytotoxic agentsin the adjuvant setting as well as in the neoadjuvant setting whenadministered agents such as 5-fluorouracil, doxorubicin andcyclophosphamide (Rouzier et al. 2005). Other agents that have shownsome efficacy include platinum based compounds such as cisplatin andanti-tubulin compounds such as taxanes (Santana-Davila and Perez, 2010).

As mentioned above, there are no specific targets for TNBC but this hasnot impeded the trial of target agents such as the inhibition of Poly[ADP-ribose] polymerase 1 (PARP1). PARP1 is an enzyme that participatesin the repair of DNA single-strand breaks by associating with corruptedDNA strands and mediating the recruitment of enzymes needed to repairsingle-strand breaks (de Ruijter et al., 2011). Thus the strategy hasbeen to inhibit PARP1 activity as a means of allowing cancer cells toaccumulate more DNA single-strand breaks, which ultimately leads togenetic instability, mitotic arrest and apoptosis. Promising clinicalresults were achieved in patients that showed mutations in BRCA1 and/orBRCA2, important mediators of genetic maintenance and homologousrecombination required for proper cell division. Indeed, patients withBRCA1 mutations, which are presumably deficient in these geneticstability pathways, showed greater response to PARP1 inhibitors comparedwith those who were wild type for BRCA1 (Fong et al., 2009). It is clearthat targeting PARP1 in TNBC patients who are carriers of BRCA mutationrepresents a promising strategy. The combination of ER/PgR/HER2 statuswith that of the genetic profile of the BRCA1/2 genes might offer thebest characterization for deciding the proper treatment options for TNBCpatients.

Other strategies also examined the use of EGFR inhibitors, either asmonoclonal antibodies or small molecule inhibitors or anti-angiogeniccompounds to target VEGF. Several clinical trials have evaluated theefficacy of these compounds but none of them have shown significantresponse when administered alone. However, mild efficacy was observed inpatients treated with these inhibitors in combination with othercytotoxic agents (Santana-Davila and Perez, 2010).

Notwithstanding the recent advances in the understanding and thetreatment for breast cancer, the use of chemotherapy is invariablyassociated with severe adverse reactions, which limit their use.Consequently, the need for more specific strategies such as combiningantigen tissue specificity with the selectivity of monoclonal antibodiesshould permit a significant reduction in off-target-associated sideeffects. There are no TNBC specific antigens that are currently underinvestigation as therapeutic targets for monoclonal antibodies. Thus,TNBC patients have little options because of the inability to target aspecific marker of protein that is expressed in these tumors. There areurgent needs to identify new proteins expressed in TNBC for applicationsas new diagnostic markers and novel targeted therapies.

Kidney associated antigen 1 (KAAG1), the protein sequence which isidentified herein as SEQ ID NO.:2, was originally cloned from a cDNAlibrary derived from a histocompatibility leukocyte antigen-B7 renalcarcinoma cell line as an antigenic peptide presented to cytotoxic Tlymphocytes (Van den Eynde et al., 1999; Genebank accesssion no. Q9UBP8,the cDNA sequence is represented by nucleotides 738-992 of SEQ IDNO.:1). The locus containing KAAG1 was found to encode two genestranscribed in both directions on opposite strands. The sense strand wasfound to encode a transcript that encodes a protein termed DCDC2.Expression studies by these authors found that the KAAG1 antisensetranscript was tumor specific and exhibited very little expression innormal tissues whereas the DCDC2 sense transcript was ubiquitouslyexpressed (Van den Eynde et al., 1999). The expression of the KAAG1transcript in cancer, and in particular ovarian cancer, renal cancer,lung cancer, colon cancer, breast cancer and melanoma was disclosed ininternational application No. PCT/CA2007/001134 published on Dec. 27,2007 under No. WO 2007/147265. Van den Eynde et al., also observed RNAexpression in renal carcinomas, colorectal carcinomas, melanomas,sarcomas, leukemias, brain tumors, thyroid tumors, mammary carcinomas,prostatic carcinomas, oesophageal carcinomas, bladder tumor, lungcarcinomas and head and neck tumors. Recently, strong genetic evidenceobtained through linkage disequilibrium studies found that theVMP/DCDC2/KAAG1 locus was associated with dyslexia (Schumacher et al.,2006; Cope et al., 2005). One of these reports pointed to the DCDC2marker as the culprit in dyslexic patients since the function of thisprotein in cortical neuron migration was in accordance with symptoms ofthese patients who often display abnormal neuronal migration andmaturation (Schumacher et al., 2006).

The Applicant has obtained a panel of antibodies and antigen bindingfragment that bind to the KAAG1 protein. These antibodies or antigenbinding fragments were shown to target three regions of the protein;amino acids 1 to 35, amino acids 36 to 60 amino acids 61 to 84. TheApplicant found that antibodies targeting a region between amino acids30 to 84 were the most advantageous for therapeutic purposes as theyrecognized KAAG1 located at the surface of tumor cells. The Applicanthas shown that some of these antibodies and antigen binding fragmentscan mediate antibody-dependent cell cytotoxicity and/or are internalizedby tumor cells, which makes them good candidates to deliver a payload totumor cells. The Applicant has also generated chimeric and humanizedantibodies based on selected antibody candidates and has shown thatthese antibodies can inhibit tumor cell formation and invasion (seePCT/CA2009/001586 published on Jun. 3, 2010 under No. WO2010/060186 andPCT/CA2010/001785 published on May 12, 2011 under No. WO2011/054112).Finally, the Applicant found that these antibodies could be used for thetreatment and diagnosis of ovarian cancer, skin cancer, renal cancer,colorectal cancer, sarcoma, leukemia, brain tumor, thyroid tumor, breastcancer, prostate cancer, oesophageal tumor, bladder tumor, lung tumorand head and neck tumor and metastatic form of these cancers.

The Applicant has now come to the unexpected discovery that breastcancer cells lacking ER protein expression, PgR protein expressionand/or showing absence of HER2 protein over-expression (i.e.,triple-negative breast cancer cells, basal-like) can be efficientlytargeted with an antibody or antigen binding fragment that specificallybinds to KAAG1. Anti-KAAG1 antibodies may thus be used for the,detection and therapeutic treatment of breast cancer cells that arenegative for at least one of these markers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is an amino acid sequence alignment of the 3A4 variable domainsof the murine and humanized light chains. The light chain has twohumanized variants (Lh1 an Lh2). The CDRs are shown in bold and indictedby CDRL1, CDRL2 and CDRL3. Back mutations in the human framework regionsthat are murine amino acids are underlined in the humanized sequences.

FIG. 1b is an amino acid sequence alignment of the 3A4 variable domainsof the murine and humanized heavy chains. The heavy chain has fourhumanized variants (Hh1 to Hh4). The CDRs are shown in bold and indictedby CDRH1, CDRH2 and CDRH3. Back mutations in the human framework regionsthat are murine amino acids are underlined in the humanized sequences.

FIG. 2a is an alignment of murine 3A4 light chain variable region (SEQID NO.:4) with a light chain variable region variant (SEQ ID NO.:33)using the ClustalW2 program (Larkin M. A., et al., (2007) ClustalW andClustalX version 2. Bioinformatics 2007 23(21): 2947-2948) where an “*”(asterisk) indicates positions which have a single, fully conservedresidue, wherein “:” (colon) indicates conservation between groups ofstrongly similar properties—scoring>0.5 in the Gonnet PAM 250 matrix andwhere “.” (period) indicates conservation between groups of weaklysimilar properties—scoring=<0.5 in the Gonnet PAM 250 matrix.

FIG. 2b is an alignment of murine 3A4 heavy chain variable region (SEQID NO.:2) with a light chain variable region variant (SEQ ID NO.:38)using the ClustalW2 program (Larkin M. A., et al., (2007) ClustalW andClustalX version 2. Bioinformatics 2007 23(21): 2947-2948) where an “*”(asterisk) indicates positions which have a single, fully conservedresidue, wherein “:” (colon) indicates conservation between groups ofstrongly similar properties—scoring>0.5 in the Gonnet PAM 250 matrix andwhere “.” (period) indicates conservation between groups of weaklysimilar properties—scoring=<0.5 in the Gonnet PAM 250 matrix.

FIG. 3a represents plasmid map of pKCR5-3A4-HC-Variant 1. The heavychains of the humanized 3A4 variants were cloned in the same manner intothe HindIII site of pK-CR5. Consequently the resulting plasmids areidentical to pKCR5-3A4-HC variant 1 except for the sequence of the heavychain immunoglobulin variable domain.

FIG. 3b represents plasmid map of pMPG-CR5-3A4-LC-Variant 1. The lightchains of the humanized variants 1 and 2 of 3A4 antibody were cloned inthe same manner into the BamHI site of pMPG-CR5. Consequently, theresulting plasmid is identical to pMPG-CR5-3A4-LC-Variant 1, except forthe sequence of the light chain immunoglobulin variable domain.

FIG. 4 represents an analysis of antibody production after transienttransfection in CHO cells. Supernatant (13 days post-transfection) ofCHOcTA cells transfected with the different combinations of light andheavy chains of humanized 3A4 antibody were analyzed by western blot.Quantification of antibody produced in the supernatants was determinedafter scanning the bands of the western blot against dilution of a knownstandard (human purified IgG antibody). Mr molecular weight marker(kDa).

FIG. 5 is a graph of a Superdex G75 gel filtration of recombinant KAAG1sample. KAAG1 was injected over the gel filtration and separated at 0.4ml/min. The largest peak between fractions 15-19.

FIG. 6 is a Table listing the rate and affinity constants for the murineand humanized variants of the 3A4 antibody.

FIG. 7a is an histogram illustrating the association rates (K_(a)) ofthe humanized antibodies.

FIG. 7b is an histogram illustrating the dissociation rates (K_(d)) ofthe humanized antibodies.

FIG. 7c is an histogram illustrating the affinity constants (K_(D)) ofthe humanized antibodies.

FIG. 8a illustrates humanized 3A4 variants binding to KAAG1 in an ELISA.This figure shows the comparative binding of 3A4 humanized antibodyvariants and the murine 3A4. Concentration-dependent binding profiles ofthe humanized heavy chains (Hh1, Hh2, Hh3 and Hh4) assembled with theLh1 light chain variant.

FIG. 8b illustrates humanized 3A4 variants binding to KAAG1 in an ELISA.This figure shows the comparative binding of 3A4 humanized antibodyvariants and the murine 3A4. Concentration-dependent binding profiles ofthe humanized heavy chains (Hh1, Hh2, Hh3 and Hh4) assembled with theLh2 light chain variant.

FIG. 9 illustrates humanized 3A4 variants binding to KAAG1 on thesurface of cancer cells. This illustration shows the comparative bindingactivity of the humanized and the murine 3A4 antibodies on theunpermeabilized SKOV-3 ovarian cancer cells.

FIG. 10 shows a scan of a tissue microarray containing 139 biopsysamples obtained from breast cancer patients. The samples were blottedwith the 3A4 anti-KAAG1 antibody and showed that the vast majority ofthe breast tumors expressed very high level of KAAG1 antigen. Theconfirmed TNBC samples are marked with an asterisk.

FIG. 11 shows the results of flow cytometry performed using MDA-MB-231,MDA-MB-436, MDA-MB-468, BT-20, BT-549, T47D, MCF-7 and 293-6E cell linesincubated with the 3A4 anti-KAAG1 antibody (blue bars of the histogram)compared with a control IgG (red bars). This is a representative resultsfrom an experiment that was performed in triplicate. The TNBC cell linesare marked with an asterisk.

FIG. 12 represents the detection of the KAAG1 antigen on the surface ofMDA-MB-231 cells by flow cytometry with the 3A4 anti-KAAG1 antibody. Thefluorescence signal decreases with time when the cells were incubated at37° C., which suggests that the KAAG1/antibody complex was internalizedduring the incubation when the cells were incubated with 3A4.

FIG. 13 represents the detection of the KAAG1 antigen on the surface ofMDA-MB-436 cells by flow cytometry with the 3A4 anti-KAAG1 antibody. Thefluorescence signal decreases with time when the cells were incubated at37° C., which suggests that the KAAG1/antibody complex was internalizedduring the incubation when the cells were incubated with 3A4.

FIG. 14 represents the detection of the KAAG1 antigen on the surface ofBT-20 cells by flow cytometry with the 3A4 anti-KAAG1 antibody. Thefluorescence signal decreases with time when the cells were incubated at37° C., which suggests that the KAAG1/antibody complex was internalizedduring the incubation when the cells were incubated with 3A4.

FIG. 15 represents the detection of the KAAG1 antigen on the surface ofT47D cells by flow cytometry with the 3A4 anti-KAAG1 antibody. Thefluorescence signal decreases with time when the cells were incubated at37° C., which suggests that the KAAG1/antibody complex was internalizedduring the incubation when the cells were incubated with 3A4.

FIG. 16 represents immunofluorescence data performed on live MDA-MB-231cells with the 3A4 anti-KAAG1 antibody and the anti-LAMP1 antibody. Theimmunofluorescence signal associated with the anti-KAAG1 antibody isshown in the left panel, the immunofluorescence signal associated LAMP1is shown in the middle panel and the merging of both images is shown inthe right panel. These data illustrates the co-localization of KAAG1 andLAMP1 near the peri-nuclear area.

FIG. 17 represents immunofluorescence data performed on live MDA-MB-231cells with the 3A4 anti-KAAG1 antibody and the anti-LAMP1 antibody. Theimmunofluorescence signal associated with the anti-KAAG1 antibody isshown in the left panel, the immunofluorescence signal associated LAMP1is shown in the middle panel and the merging of both images is shown inthe right panel. These data illustrates the localization of KAAG1 withLAMP1 a marker of late endosomes/lysosomes.

SUMMARY OF THE INVENTION

The present invention provides a method of treating or detecting canceror cancer cells (in vitro or in vivo) in an individual in need.

In accordance with the present invention, methods of treatment ordetection may be carried out with an antibody capable of binding toKAAG1 or an antigen binding fragment thereof.

The individual in need may comprise, for example, an individual havingor suspected of having cancer. Such individual may have a cancer orcancer cells originating from a breast carcinoma.

The cancer or cancer cells may more particularly originate from a breastcarcinoma characterized as being triple-negative or basal-like.

Therefore, the individuals who may benefit from methods of treatment ordetection described herein may include those suffering from breastcarcinoma.

The breast carcinoma may comprise tumors cells showing a decrease or alost in the expression of the estrogen receptor.

The breast carcinoma may comprise tumor cells showing a decrease or alost in the expression of the progesterone receptor.

The breast carcinoma may comprise tumor cells showing a decrease or alost in the expression of Her2.

The breast carcinoma may comprise tumor cells showing a decrease or alost in Her2 overexpression.

More particularly, the breast carcinoma may comprise tumor cells showingeither 1) a decrease or a loss in expression of the estrogen receptorand the progesterone receptor, 2) a decrease or a loss in expression ofthe estrogen receptor and a decrease or a loss of Her2 overexpression,3) a decrease or a loss in expression of the progesterone receptor and adecrease or a loss of Her2 overexpression or 4) a decrease or a loss inexpression of the estrogen receptor, a decrease or a loss in expressionof the progesterone receptor and a decrease or a loss of Her2overexpression.

Even more particularly, the breast carcinoma may comprise tumor cellsshowing either 1) a loss in expression of the estrogen receptor and theprogesterone receptor, 2) a loss in expression of the estrogen receptorand a loss of Her2 expression, 3) a loss in expression of theprogesterone receptor and a loss of Her2 expression or 4) a loss inexpression of the estrogen receptor, a loss in expression of theprogesterone receptor and a loss of Her2 expression.

In accordance with the present invention, the individual may carrybreast cancer cells that are characterized as being triple-negative ormay have a tumor categorized as being a triple-negative breast cancer.

In accordance with the present invention, the individual may carrybreast cancer cells that are characterized as basal-like, or may have atumor categorized as being a basal-like breast cancer.

Other individuals who would benefit from treatment with an anti-KAAG1include those having carcinoma comprising tumors cells exhibiting anepithelial-to-mesenchymal transition (EMT) phenotype.

Commonly used molecular markers of EMT include, for example, a reducedexpression of E-cadherin, cytokeratin and β-catenin (in the membrane)and/or an increased expression of Snail, Slug, Twist, ZEB1, ZEB2,N-cadherin, vimentin, α-smooth muscle actin, matrix metalloproteinasesetc. (see for example, Kalluri and Weinberg, The Journal of ClinicalInvestigation, 119(6), p 1420-1428; 2009; Fassina et al., ModernPathology, 25; p 86-99; 2012; Lee et al., JCB; 172; p 973-981; 2006). AnEMT phenotype may also be distinguished by an increased capacity formigration, invasion of by resistance to anoikis/apoptosis. Cells thatare undergoing epithelial-to-mesenchymal transition may thus be detectedby a reduction of epithelial markers and apparition of mesenchymalmarkers or EMT phenotypes.

In accordance with the present invention, the method may thus comprise,for example, administering an antibody or antigen binding fragment whichis capable of specific binding to KAAG1 to an individual in need. Theindividual in need is preferentially selected on the basis of theirtumor lacking ER expression, PgR expression and/or by the absence ofHER2 protein over-expression. Clinical testing for these markers isusually performed using histopathologic methods (immunohistochemistry,FISH, etc.) and/or by gene expression studies (see for example Dent etal, 2007, Bernstein and Lacey, 2011). The individual in need may thus bean individual who has received a diagnosis of triple-negative breastcancer or basal-like breast cancer. The individual in need may be anindividual which is unresponsive to hormonal therapy and/or totranstuzumab therapy (or other anti-Her2 antibodies). Alternatively, theindividual in need may be an individual carrying tumor cells that havethe ability of undergoing epithelial-to-mesenchymal transition or thathave acquired a mesenchymal phenotype.

The present invention thus provides a method of treating triple-negativebreast cancer or basal-like breast cancer by administering an inhibitorof KAAG1 activity or expression to an individual in need.

In accordance with the present invention, the KAAG1 inhibitor may thuscomprise an antibody described herein or an antigen binding fragmentthereof.

Also in accordance with the present invention, the KAAG1 inhibitor maycomprise a nucleotide sequence complementary to SEQ ID NO.:1 or to afragment thereof. More particularly, the KAAG1 inhibitor may comprise anucleotide sequence complementary to nucleotides 738 to 992(inclusively) of SEQ ID NO.:1 or to a fragment thereof. For example, theinhibitor may include at least 10 consecutive nucleotides (at least 15,at least 20) which are complementary to SEQ ID NO.:1 or to nucleotides738 to 992 (inclusively) of SEQ ID NO.:1. More particular type of KAAG1inhibitor includes a siRNA which inhibit expression of SEQ ID NO.:1.

Suitable antibodies or antigen binding fragments include those that arecapable of binding to KAAG1 at the surface of tumor cells. Suchantibodies or antigen binding fragments thereof may preferentially bindan epitope included within amino acids 30 to 84 of KAAG1 inclusively.

Alternatively such antibodies or antigen binding fragments thereof maybind an epitope located within amino acids 36 to 60 (inclusively) orwithin amino acids 61 to 84 (inclusively) of KAAG1.

The epitope may particularly be located or comprised within amino acids50 to 70, 50 to 65, 51 to 65, 52 to 65, 53 to 65, 54 to 65, 54 to 64, 54to 63, 54 to 62, 54 to 61, 54 to 60, 50 to 62; 50 to 61, or 50 to 60(inclusively or exclusively).

In accordance with an embodiment of the invention, the antibody orantigen binding fragment may bind an epitope comprised within aminoacids 50 to 70 of KAAG1.

In a further embodiment of the invention, the antibody or antigenbinding fragment may bind an epitope comprised within amino acids 50 to62 of KAAG1.

In yet a further embodiment, the antibody or antigen binding fragmentmay bind an epitope comprised within amino acids 54 to 65 of KAAG1.

Suitable antibodies for therapeutic treatment include for example, thosewhich mediate antibody-dependent cell cytotoxicity.

Other even more suitable antibodies for therapeutic treatment includethose that are conjugated with a therapeutic moiety.

In accordance with the present invention, the antibody may be, forexample, a monoclonal antibody, a chimeric antibody, a humanizedantibody a human antibody or an antigen binding fragment thereof.

DETAILED DESCRIPTION OF THE INVENTION Method of Treatment

As indicated herein, the present invention encompass administering anantibody or antigen binding fragment to an individual having a breastcancer characterized as being “triple negative breast cancer” or“basal-like breast cancer”.

Classification of breast cancer subtypes as being “triple negativebreast cancer” or “basal-like breast cancer” is known in the art (seefor example, Foulkes et al., N. Engl. J. Med., 2010; 363:1938-1948) andincludes, for example, the following definitions:

“Basal-like breast cancer”, may include for example, a subtype of breastcancer comprising a heterogenous group of tumors characterized by theabsence of or low levels of expression of estrogen receptors, very lowprevalence of Her2 overexpression and expression of genes usually foundin the basal or myoepithelial cells of the human breast. Such expressionmay be determined by microarray analysis.

“Triple-negative breast cancer”, may include for example, a tumorcharacterized by lack of estrogen receptor (ER), progesterone receptor(PR) and Her2 expression. Some investigators accept tumors as beingnegative for expression of ER or PR only if less than 1% of the cellsare positive for ER or PR expression; others consider tumors to benegative for ER or PR expression when up to 10% of cells are positivefor expression. Different definitions of HER2-negativity have been used.The two most frequently adopted include tumors with immunohistochemicalscores of 0/1+ or 2+ that are lacking HER2 gene amplification after insitu hybridization. Such expression may be especially determined byimmunohistochemical staining.

In accordance with the present invention, the method of treatmentincludes administering a KAAG1 inhibitor to an individual in need. SuchKAAG1 inhibitor includes, for example, an antibody or antigen bindingfragment thereof which specifically binds to KAAG1.

It is likely that the most potent antibodies or antigen bindingfragments may be those having a high affinity for KAAG1. It is alsolikely that the most potent antibodies or antigen binding fragments maybe those that are internalized within a cells compartment such as, forexample, a lysosome or an endosome.

As such, the present invention especially encompasses antibodies orantigen binding fragments having a high affinity for KAAG1.

Suitable antibodies or antigen binding fragments include those that arecapable of binding to KAAG1 at the surface of tumor cells with a highaffinity. Such high affinity antibodies or antigen binding fragmentsthereof may preferentially bind an epitope included within amino acids30 to 84 of KAAG1 inclusively.

Alternatively such high affinity antibodies or antigen binding fragmentsthereof may bind an epitope located within amino acids 36 to 60(inclusively) or within amino acids 61 to 84 (inclusively) of KAAG1.

The high affinity antibodies or antigen binding fragments may bind, forexample, an epitope may particularly be located or comprised withinamino acids 50 to 70, 50 to 65, 51 to 65, 52 to 65, 53 to 65, 54 to 65,54 to 64, 54 to 63, 54 to 62, 54 to 61, 54 to 60, 50 to 62; 50 to 61, or50 to 60 (inclusively or exclusively).

In accordance with an embodiment of the invention, the high affinityantibody or antigen binding fragment may bind an epitope comprisedwithin amino acids 50 to 70 of KAAG1.

In a further embodiment of the invention, the high affinity antibody orantigen binding fragment may bind an epitope comprised within aminoacids 50 to 62 of KAAG1.

In yet a further embodiment, the high affinity antibody or antigenbinding fragment may bind an epitope comprised within amino acids 54 to65 of KAAG1.

Preferred antibodies including high affinity antibodies are those thanmay be internalized in a cell or cell compartment (e.g., lysosomes orendosomes). The ability of antibodies to be internalized may bedetermined by method known in the art such as for example and withoutlimitation, by immunofluorescence studies similar to those performedherein.

Antibodies having CDRs identical to those of the 3A4 antibodies areparticularly encompassed by the present invention. As such, antibodieshaving a light chain variable region and/or heavy chain variable regionconsensus sequences set forth in any of SEQ ID NOs.:186 to 188 and 191to 193 and specific sequences set forth in SEQ ID No.:46, 48, 189, 190,or 194 to 198 are encompassed by the present invention. Among those,antibodies having a light chain variable region and/or heavy chainvariable region consensus sequences set forth in any of SEQ ID NO.: 188and 196 or specific sequences set forth in SEQ ID NO.:46, 48, 189, 190,or 194 to 198 are particularly contemplated.

The antibodies or antigen binding fragments thereof may preferably beconjugated with a therapeutic moiety.

The antibodies or antigen binding fragments thereof, may have a humanconstant region. Preferably the antibodies or antigen binding fragmentsthereof may have a human IgG1 constant region. Alternatively, theantibodies or antigen binding fragments thereof may have an IgG2constant region.

The method of the present invention may also include administering aKAAG1 inhibitor such as an antibody (e.g., conjugated with a therapeuticmoiety) or antigen binding fragment in combination with an anticanceragent such as for example, a small molecule drug, an antibody or antigenbinding fragment binding to a target other than KAAG1, achemotherapeutic or a cytotoxic agent. Example of anticancer agent thatcould be administered with the KAAG1 inhibitor may include for example,doxorubicin, taxanes, anti-angiogenic agents, platinum salts, PARPinhibitors.

Other methods of treatment encompassed by the present invention includeadministering other types of KAAG1 inhibitors such as antisense-basedtherapeutics (siRNA, antisenses, ribozymes, etc.).

Antibodies and Antigen Binding Fragments that Binds to KAAG1

The term “antibody or antigen binding fragment” or similar terms such as“antibodies and antigen binding fragments” encompasses, for example“variant antibody or antigen binding fragment” such as, for example,“humanized antibody or antigen binding fragment”.

The term “antibody” refers to intact antibody, monoclonal or polyclonalantibodies. The term “antibody” also encompasses multispecificantibodies such as bispecific antibodies. Human antibodies are usuallymade of two light chains and two heavy chains each comprising variableregions and constant regions. The light chain variable region comprises3 CDRs, identified herein as CDRL1, CDRL2 and CDRL3 flanked by frameworkregions. The heavy chain variable region comprises 3 CDRs, identifiedherein as CDRH1, CDRH2 and CDRH3 flanked by framework regions.

The term “antigen-binding fragment”, as used herein, refers to one ormore fragments of an antibody that retain the ability to bind to anantigen (e.g., KAAG1, secreted form of KAAG1 or variants thereof). Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of an intact antibody. Examples of bindingfragments encompassed within the term “antigen-binding fragment” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe V_(L), V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the V_(H)and C_(H1) domains; (iv) a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,(1989) Nature 341:544-546), which consists of a V_(H) domain; and (vi)an isolated complementarity determining region (CDR), e.g., V_(H) CDR3.Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single polypeptide chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen-bindingfragment” of an antibody. Furthermore, the antigen-binding fragmentsinclude binding-domain immunoglobulin fusion proteins comprising (i) abinding domain polypeptide (such as a heavy chain variable region, alight chain variable region, or a heavy chain variable region fused to alight chain variable region via a linker peptide) that is fused to animmunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavychain CH2 constant region fused to the hinge region, and (iii) animmunoglobulin heavy chain CH3 constant region fused to the CH2 constantregion. The hinge region may be modified by replacing one or morecysteine residues with serine residues so as to prevent dimerization.Such binding-domain immunoglobulin fusion proteins are further disclosedin US 2003/0118592 and US 2003/0133939. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

A typical antigen binding site is comprised of the variable regionsformed by the pairing of a light chain immunoglobulin and a heavy chainimmunoglobulin. The structure of the antibody variable regions is veryconsistent and exhibits very similar structures. These variable regionsare typically comprised of relatively homologous framework regions (FR)interspaced with three hypervariable regions termed ComplementarityDetermining Regions (CDRs). The overall binding activity of the antigenbinding fragment is often dictated by the sequence of the CDRs. The FRsoften play a role in the proper positioning and alignment in threedimensions of the CDRs for optimal antigen binding.

As used herein the term “high affinity” refers to an affinity of 10 nMor less. The term “high affinity” especially includes antibodies havingan affinity of 5 nM or less. The term “high affinity” even moreparticularly includes antibodies having an affinity of 1 nM or less, or0.1 nM or less.

Antibodies and/or antigen binding fragments of the present invention mayoriginate, for example, from a mouse, a rat or any other mammal or fromother sources such as through recombinant DNA technologies.

An-KAAG1 antibodies were initially isolated from Fab libraries for theirspecificity towards the antigen of interest. Exemplary methods on how toconvert Fab into full immunoglobulins are provided herein.

The variable regions described herein may be fused with constant regionsof a desired species thereby allowing recognition of the antibody byeffector cells of the desired species. The constant region mayoriginate, for example, from an IgG1, IgG2, IgG3, or IgG4 subtype.Cloning or synthesizing a constant region in frame with a variableregion is well within the scope of a person of skill in the art and maybe performed, for example, by recombinant DNA technology.

In certain embodiments of the present invention, antibodies that bind toKAAG1 may be of the IgG1, IgG2, IgG3, or IgG4 subtype. More specificembodiments of the invention relates to an antibody of the IgG1 subtypeor especially human IgG1 subtype. Other specific embodiments of theinvention relates to an antibody of the IgG2 subtype or especially ofthe human IgG2 subtype.

The antibody may be a humanized antibody of the IgG1 subtype subtype orespecially human IgG1 subtype. Alternatively, the antibody may be ahumanized antibody of the IgG2 subtype or especially of the human IgG2subtype.

The antibody may be, for example, biologically active in mediatingantibody-dependent cellular cytotoxicity (ADCC), complement-mediatedcytotoxicity (CMC), or associated with immune complexes. The typicalADCC involves activation of natural killer (NK) cells and is reliant onthe recognition of antibody-coated cells by Fc receptors on the surfaceof the NK cells. The Fc receptors recognize the Fc domain of antibodiessuch as is present on IgG1, which bind to the surface of a target cell,in particular a cancerous cell that expresses an antigen, such as KAAG1.Once bound to the Fc receptor of IgG the NK cell releases cytokines andcytotoxic granules that enter the target cell and promote cell death bytriggering apoptosis.

The present invention described a collection of antibodies that bind toKAAG1 or to a KAAG1 variant. In certain embodiments, the antibodies maybe selected from the group consisting of polyclonal antibodies,monoclonal antibodies such as chimeric or humanized antibodies, antibodyfragments such as antigen binding fragments, single chain antibodies,domain antibodies, and polypeptides with an antigen binding region.

In an aspect of the invention, the isolated antibody or antigen bindingfragment of the present invention may be capable of inducing killing(elimination, destruction, lysis) of KAAG1-expressing tumor cells orKAAG1 variant-expressing tumor cells (e.g., in an ADCC-dependentmanner).

In a further aspect of the invention, the isolated antibody or antigenbinding fragment of the present invention may especially becharacterized by its capacity of reducing spreading of tumor cellsexpressing KAAG1 or a KAAG1 variant.

In an additional aspect of the invention, the isolated antibody orantigen binding fragment of the present invention may be characterizedby its capacity of decreasing or impairing formation of tumorsexpressing KAAG1 or a KAAG1 variant.

In an exemplary embodiment of the invention, the isolated antibody orantigen binding fragment may comprise amino acids of a constant region,which may originate, for example, from a human antibody.

In another exemplary embodiment of the invention, the isolated antibodyor antigen binding fragment may comprise framework amino acids of ahuman antibody.

Without being limited to the exemplary embodiments presented herein, theApplicant as generated specific antibodies and antigen binding fragmentsthat may be useful for the purposes described herein.

The following is a list of antibodies that were generated and shown tobind in a specific manner to KAAG1; 3D3, 3A4, 3C4, 3G10, 3A2, 3F6, 3E8,3E10, 3A9, 3B1, 3G5, 3B2, 3B8, 3G8, 3F7, 3E9, 3G12, 3C3, 3E12, 4A2,3F10, 3F4, 3611, 3D1, 3C2, 3E6 and 3H3. Sequences of the antibody lightchain or heavy chain, variable regions or complementary determiningregions (CDRs) are available in international application No.PCT/CA2009/001586 published on Jun. 3, 2010 under No. WO2010/060186A8,in international application No. PCT/CA2010/001795 published on May 12,2011 under No. WO2011/054112A1 or in international application No.PCT/CA2012/000296 published on Oct. 4, 2012 under No. WO2012/129668A1.

In most instances, the sequence of the CDRs has been provided separatelyor is shown in bold herein.

Amongst, these antibodies, the 3D3, 3A4, 3G10 and 3C4 were selected forin vitro and/or in vivo biological testing. The 3A4 antibody appeared tohave the best characteristics. Based on our experiments, the 3A4antibody when conjugated with a therapeutic moiety (e.g. a cytotoxicagent) is more effective in killing cancer cells than its non-conjugatedversion.

In an exemplary embodiment, the antibody or antigen binding fragment maycomprise any individual CDR or a combination of CDR1, CDR2 and/or CDR3of the light chain variable region. The CDR3 may more particularly beselected. Combination may include for example, CDRL1 and CDRL3; CDRL1and CDRL2; CDRL2 and CDRL3 and; CDRL1, CDRL2 and CDRL3.

In another exemplary embodiment, the antibody or antigen bindingfragment may comprise any individual CDR or a combination of CDR1, CDR2and/or CDR3 of the heavy chain variable region. The CDR3 may moreparticularly be selected. Combination may include for example, CDRH1 andCDRH3; CDRH1 and CDRH2; CDRH2 and CDRH3 and; CDRH1, CDRH2 and CDRH3.

In accordance with the present invention, the antibody or antigenbinding fragment may comprise at least two CDRs of a CDRL1, a CDRL2 or aCDRL3.

Also in accordance with the present invention, the antibody or antigenbinding fragment may comprise one CDRL1, one CDRL2 and one CDRL3.

Further in accordance with the present invention, the antibody orantigen binding fragment may comprise:

-   -   a. At least two CDRs of a CDRL1, CDRL2 or CDRL3 and;    -   b. At least two CDRs of a CDRH1, one CDRH2 or one CDRH3.

The antibody or antigen binding fragment may more preferably compriseone CDRL1, one CDRL2 and one CDRL3.

The antibody or antigen binding fragment may also more preferablycomprise one CDRH1, one CDRH2 and one CDRH3.

When only one of the light chain variable region or the heavy chainvariable region is available, an antibody or antigen-binding fragmentmay be reconstituted by screening a library of complementary variableregions using methods known in the art (Portolano et al. The Journal ofImmunology (1993) 150:880-887, Clarkson et al., Nature (1991)352:624-628).

Exemplary embodiments of the present invention encompass antibodies orantigen binding fragments having the CDRs of the light chain and/orheavy chains of the 3D3, 3A4, 3C4, 3G10, 3A2, 3F6, 3E8, 3E10, 3A9, 3B1,3G5, 3B2, 368, 3G8, 3F7, 3E9, 3G12, 3C3, 3E12, 4A2, 3F10, 3F4, 3611,3D1, 3C2, 3E6 or 3H3 antibodies. More particular embodiments of theinvention include antibodies or antigen binding fragments having theCDRs of the light chain and/or heavy chains of the 3D3, 3A4, 3C4 or 3G10antibodies. Even more particular embodiments of the invention includeantibodies or antigen binding fragments having the CDRs of the lightchain and/or heavy chains of the 3A4 antibody. The invention thusencompassed any monoclonal, chimeric, human, or humanized antibodycomprising one or more CDRs of the 3A4 antibody.

Antibodies or antigen binding fragments that may be used in methods ofthe present invention, include those having CDRs of the 3A4 antibody andmay comprise, for example, a CDRH1 as set forth in SEQ ID NO.:49, aCDRH2 as set forth in SEQ ID NO.:50 or in SEQ ID NO.:212, a CDRH3 as setforth in SEQ ID NO.:51, a CDRL1 as set forth in SEQ ID NO.: 52, a CDRL2as set forth in SEQ ID NO.:53 and a CDRL3 as set forth in SEQ ID NO.:54.

The present invention therefore encompass, antibodies and antigenbinding fragment which are capable of specific binding to KAAG1 andwhich may comprise sequences selected from the group consisting of:

-   -   a. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:16 and/or the 3CDRs of a heavy chain variable region defined        in SEQ ID NO.:18,    -   b. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:20 and/or the 3CDRs of a heavy chain variable region defined        in SEQ ID NO.:22;    -   c. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:24 and/or the 3CDRs of a heavy chain variable region defined        in SEQ ID NO.:26;    -   d. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:48 and/or the 3CDRs of a heavy chain variable region defined        in SEQ ID NO.:46;    -   e. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:103 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:126,    -   f. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:104 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:127,    -   g. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:105 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:128,    -   h. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:106 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:145,    -   i. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:107 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:129,    -   j. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:108 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:130,    -   k. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:109 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:141,    -   l. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:110 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:131,    -   m. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:111 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:134,    -   n. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:112 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:135,    -   o. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:113 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:136,    -   p. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:114 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:133,    -   q. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:115 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:140,    -   r. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:116 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:137,    -   s. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:117 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:144,    -   t. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:118 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:139,    -   u. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:119 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:132,    -   v. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:120 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:142,    -   w. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:121 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:138,    -   x. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:122 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:146,    -   y. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:123 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:153,    -   z. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:124 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:143,    -   aa. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:189 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:194,    -   bb. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:189 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:195,    -   cc. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:189 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:196,    -   dd. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:189 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:197,    -   ee. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:190 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:194,    -   ff. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:190 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:195,    -   gg. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:190 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:196, or    -   hh. the 3CDRs of a light chain variable region defined in SEQ ID        NO.:190 and/or the 3CDRs of a heavy chain variable region        defined in SEQ ID NO.:197.

Other exemplary embodiments of the invention encompass antibodies orantigen binding fragments having the light chain and/or heavy chains ofthe 3D3, 3A4, 3C4, 3G10, 3A2, 3F6, 3E8, 3E10, 3A9, 361, 3G5, 3B2, 368,3G8, 3F7, 3E9, 3G12, 3C3, 3E12, 4A2, 3F10, 3F4, 3611, 3D1, 3C2, 3E6 or3H3 antibodies. More particular embodiments of the invention includeantibodies or antigen binding fragments having the light chain and/orheavy chains of the 3D3, 3A4, 3C4 or 3G10 antibodies. Even moreparticular embodiments of the invention include antibodies or antigenbinding fragments having the light chain and/or heavy chains of the 3A4antibody (humanized and non-humanized).

The present invention therefore encompass, antibodies and antigenbinding fragment which are capable of specific binding to KAAG1 andwhich may comprise sequences selected from the group consisting of:

-   -   a. the light chain variable region defined in SEQ ID NO.:16        (encoded by SEQ ID NO.:15) and/or the heavy chain variable        region defined in SEQ ID NO.:18 (encoded by SEQ ID NO.:17),    -   b. the light chain variable region defined in SEQ ID NO.:20        (encoded by SEQ ID NO.:19) and/or the heavy chain variable        region defined in SEQ ID NO.:22 (encoded by SEQ ID NO.:21);    -   c. the light chain variable region defined in SEQ ID NO.:24        (encoded by SEQ ID NO.:23) and/or the heavy chain variable        region defined in SEQ ID NO.:26 (encoded by SEQ ID NO.:25);    -   d. the light chain variable region defined in SEQ ID NO.:48        and/or the heavy chain variable region defined in SEQ ID NO.:46,    -   e. the light chain variable region defined in SEQ ID NO.:103        and/or the heavy chain variable region defined in SEQ ID        NO.:126,    -   f. the light chain variable region defined in SEQ ID NO.:104        and/or the heavy chain variable region defined in SEQ ID        NO.:127,    -   g. the light chain variable region defined in SEQ ID NO.:105        and/or the heavy chain variable region defined in SEQ ID        NO.:128,    -   h. the light chain variable region defined in SEQ ID NO.:106        and/or the heavy chain variable region defined in SEQ ID        NO.:145,    -   i. the light chain variable region defined in SEQ ID NO.:107        and/or the heavy chain variable region defined in SEQ ID        NO.:129,    -   j. the light chain variable region defined in SEQ ID NO.:108        and/or the heavy chain variable region defined in SEQ ID        NO.:130,    -   k. the light chain variable region defined in SEQ ID NO.:109        and/or the heavy chain variable region defined in SEQ ID        NO.:141,    -   l. the light chain variable region defined in SEQ ID NO.:110        and/or the heavy chain variable region defined in SEQ ID        NO.:131,    -   m. the light chain variable region defined in SEQ ID NO.:111        and/or the heavy chain variable region defined in SEQ ID        NO.:134,    -   n. the light chain variable region defined in SEQ ID NO.:112        and/or the heavy chain variable region defined in SEQ ID        NO.:135,    -   o. the light chain variable region defined in SEQ ID NO.:113        and/or the heavy chain variable region defined in SEQ ID        NO.:140,    -   p. the light chain variable region defined in SEQ ID NO.:114        and/or the heavy chain variable region defined in SEQ ID        NO.:133,    -   q. the light chain variable region defined in SEQ ID NO.:115        and/or the heavy chain variable region defined in SEQ ID        NO.:140,    -   r. the light chain variable region defined in SEQ ID NO.:116        and/or the heavy chain variable region defined in SEQ ID        NO.:137,    -   s. the light chain variable region defined in SEQ ID NO.:117        and/or the heavy chain variable region defined in SEQ ID        NO.:144,    -   t. the light chain variable region defined in SEQ ID NO.:118        and/or the heavy chain variable region defined in SEQ ID        NO.:139,    -   u. the light chain variable region defined in SEQ ID NO.:119        and/or the heavy chain variable region defined in SEQ ID        NO.:132,    -   v. the light chain variable region defined in SEQ ID NO.:120        and/or the heavy chain variable region defined in SEQ ID        NO.:142,    -   w. the light chain variable region defined in SEQ ID NO.:121        and/or the heavy chain variable region defined in SEQ ID        NO.:138,    -   x. the light chain variable region defined in SEQ ID NO.:122        and/or the heavy chain variable region defined in SEQ ID        NO.:146,    -   y. the light chain variable region defined in SEQ ID NO.:123        and/or the heavy chain variable region defined in SEQ ID        NO.:147;    -   z. the light chain variable region defined in SEQ ID NO.:124        and/or the heavy chain variable region defined in SEQ ID        NO.:144;    -   aa. the light chain variable region defined in SEQ ID NO.:189        and/or the heavy chain variable region defined in SEQ ID        NO.:194,    -   bb. the light chain variable region defined in SEQ ID NO.:189        and/or the heavy chain variable region defined in SEQ ID        NO.:195,    -   cc. the light chain variable region defined in SEQ ID NO.:190        and/or the heavy chain variable region defined in SEQ ID        NO.:194,    -   dd. the light chain variable region defined in SEQ ID NO.:190        and/or the heavy chain variable region defined in SEQ ID        NO.:195,    -   ee. the light chain variable region defined in SEQ ID NO.:190        and/or the heavy chain variable region defined in SEQ ID        NO.:196, or    -   ff. the light chain variable region defined in SEQ ID NO.:190        and/or the heavy chain variable region defined in SEQ ID        NO.:197.

The framework region of the heavy and/or light chains described hereinmay be derived from one or more of the framework regions illustrated inthe antibodies described herein. The antibody or antigen bindingfragments may thus comprise one or more of the CDRs described herein(e.g., selected from the specific CDRs or consensus CDRs of SEQ IDNO.:72 to 88 or CDR variants of SEQ ID NO.:89-102) and framework regionsoriginating from those described herein. In SEQ ID Nos. 103-154, theexpected CDRs are shown in bold, while the framework regions are not.

Table 1 refers to the complete sequences of light and heavy chain ofsome of the anti-KAAG1 antibodies which were selected for biologicaltesting.

TABLE 1 Nucleotide Amino acid Antibody sequence sequence designationChain type (SEQ ID NO.:) (SEQ ID NO.:) 3D3 Light (L) 3 4 3D3 Heavy (H) 56 3G10 Light 7 8 3G10 Heavy 9 10 3C4 Light 11 12 3C4 Heavy 13 14Humanized 3D3 Light 166 Humanized 3D3 Heavy 167 Humanized 3C4 Light 170Humanized 3C4 Heavy 171 Humanized 3A4 Light (Lh1) 199 Humanized 3A4Light (Lh2) 200 Humanized 3A4 Heavy (Hh1) 202 Humanized 3A4 Heavy (Hh2)203 Humanized 3A4 Heavy (Hh3) 204 Humanized 3A4 Heavy (Hh4) 205

Epitope mapping studies revealed that the 3D3 antibody interacts with aKAAG1 epitope spanned by amino acids 36-60, inclusively. The 3G10 and3A4 antibodies interact with a KAAG1 epitope spanned by amino acids61-84, inclusively and the 3C4 antibody interacts with a KAAG1 epitopespanned by amino acids 1-35. Although, the 3G10 and 3A4 binds a similarregion, the 3G10 antibody does not bind to KAAG1 as efficiently as the3A4 antibody.

It is to be understood herein, that the light chain variable region ofthe specific combination provided above may be changed for any otherlight chain variable region. Similarly, the heavy chain variable regionof the specific combination provided above may be changed for any otherheavy chain variable region.

Sequences of light and heavy chain variable regions of selectedantibodies that bind to KAAG1 are disclosed in Table 2.

TABLE 2 Ab. Variable Nucleotide Amino acid designation region type (SEQID NO.:) (SEQ ID NO.:) 3D3 Light (VL) 15 16 3D3 Heavy (VH) 17 18 3G10Light 19 20 3G10 Heavy 21 22 3C4 Light 23 24 3C4 Heavy 25 26 3A2 Light103 3A2 Heavy 126 3E10 Light 106 3E10 Heavy 145 3G12 Light 121 3G12Heavy 138 3A4 Light 47 48 3A4 Heavy 45 46 Humanized 3D3 Light 168Humanized 3D3 Heavy 169 Humanized 3C4 Light 172 Humanized 3C4 Heavy 173Humanized 3A4 Light (Lvh1) 189 Humanized 3A4 Light (Lvh2) 190 Humanized3A4 Heavy (Hvh1) 194 Humanized 3A4 Heavy (Hvh2) 195 Humanized 3A4 Heavy(Hvh3) 197 Humanized 3A4 Heavy (Hvh4) 198

SEQ ID NOs. 103-154 correspond to the light chain and heavy chainvariable regions of other antibodies which were shown to bind KAAG1.

CDR sequence of the light and heavy chain variable regions of selectedantibodies that bind to KAAG1 are disclosed in Table 3.

TABLE 3 Ab. SEQ desig- Chain ID a.a. nation type CDR NO.: sequence 3D3Light (L) CDR L1  27 KSSQSLLNSNFQKNFLA 3D3 Light CDR L2  28 FASTRES 3D3Light CDR L3  29 QQHYSTPLT 3D3 Heavy (H) CDR H1  30 GYIFTDYEIH 3D3 HeavyCDR H2  31 VIDPETGNTA 3D3 Heavy CDR H3  32 MGYSDY 3G10 Light CDR L1  33RSSQSLLHSNGNTYLE 3G10 Light CDR L2  34 KVSNRFS 3G10 Light CDR L3  35FQGSHVPLT 3G10 Heavy CDR H1  36 GYTFTDNYMN 3G10 Heavy CDR H2  37DINPYYGTTT 3G10 Heavy CDR H3  38 ARDDWFDY 3C4 Light CDR L1  39KASQDIHNFLN 3C4 Light CDR L2  40 RANRLVD 3C4 Light CDR L3  41 LQYDEIPLT3C4 Heavy CDR H1  42 GFSITSGYGWH 3C4 Heavy CDR H2  43 YINYDGHND 3C4Heavy CDR H3 44 ASSYDGLFAY 3A2 Light CDR L1 148 KSSQSLLHSDGKTYLN 3A2Light CDR L2 149 LVSKLDS 3A2 Light CDR L3 150 WQGTHFPRT 3A2 Heavy CDR H1151 GYTFTD YNMH 3A2 Heavy CDR H2 152 YINPYNDVTE 3A2 Heavy CDR H3 153AWFGL RQ 3E10 Light CDR L1 154 RSSKSLLHSNGN TYLY 3E10 Light CDR L2 155RMSNLAS 3E10 Light CDR L3 156 MQHLEYPYT 3E10 Heavy CDR H1 157GDTFTD YYMN 3E10 Heavy CDR H2 158 DINPNYGGIT 3E10 Heavy CDR H3 159QAYYRNS DY 3G12 Light CDR L1 160 KASQDVGTAVA 3G12 Light CDR L2 161VVTSTRHT 3G12 Light CDR L3 162 QQHYSIPLT 3G12 Heavy CDR H1 163GYIFTDYEIH 3G12 Heavy CDR H2 164 VIDPETGNTA 3G12 Heavy CDR H3 165 MGYSDY3A4 Light CDR L1  52 RSSQSLLHSNGNTYLE 3A4 Light CDR L2  53 TVSNRFS 3A4Light CDR L3  54 FQGSHVPLT 3A4 Heavy CDR H1  49 GYTFTDDYMS 3A4 HeavyCDR H2  50  DINPYNGDTNYNQKFKG or or DINPYNGDTN 212 3A4 Heavy CDR H3  51DPGAMDY

Variant Antibody and Antigen Binding Fragments

The present invention also encompasses variants of the antibodies orantigen binding fragments described herein. Variant antibodies orantigen binding fragments included are those having a variation in theamino acid sequence. For example, variant antibodies or antigen bindingfragments included are those having at least one variant CDR (two,three, four, five or six variant CDRs, etc. or even twelve variantCDRs), a variant light chain variable region, a variant heavy chainvariable region, a variant light chain and/or a variant heavy chain.Variant antibodies or antigen binding fragments included in the presentinvention are those having, for example, similar or improved bindingaffinity in comparison with the original antibody or antigen bindingfragment.

As used herein the term “variant” applies to any of the sequencedescribed herein and includes for example, a variant CDR (either CDRL1,CDRL2, CDRL3, CDRH1, CDRH2 and/or CDRH3), a variant light chain variableregion, a variant heavy chain variable region, a variant light chain, avariant heavy chain, a variant antibody, a variant antigen bindingfragment and a KAAG1 variant.

The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions (CDRs), but modifications in the frameworkregion or even in the constant region are also contemplated. Exemplaryembodiments of CDR variants are provided in SEQ ID NOs.: 72-102.

Conservative substitutions may be made by exchanging an amino acid (of aCDR, variable chain, antibody, etc.) from one of the groups listed below(group 1 to 6) for another amino acid of the same group.

Other exemplary embodiments of conservative substitutions are shown inTable 1A under the heading of “preferred substitutions”. If suchsubstitutions result in a undesired property, then more substantialchanges, denominated “exemplary substitutions” in Table 1A, or asfurther described below in reference to amino acid classes, may beintroduced and the products screened.

It is known in the art that variants may be generated by substitutionalmutagenesis and retain the biological activity of the polypeptides ofthe present invention. These variants have at least one amino acidresidue in the amino acid sequence removed and a different residueinserted in its place. For example, one site of interest forsubstitutional mutagenesis may include a site in which particularresidues obtained from various species are identical. Examples ofsubstitutions identified as “conservative substitutions” are shown inTable 1A. If such substitutions result in a change not desired, thenother type of substitutions, denominated “exemplary substitutions” inTable 1A, or as further described herein in reference to amino acidclasses, are introduced and the products screened.

Substantial modifications in function or immunological identity areaccomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation. (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side chainproperties:

(group 1) hydrophobic: norleucine, methionine (Met), Alanine (Ala),Valine (Val), Leucine (Leu), Isoleucine (Ile)(group 2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine(Thr)(group 3) acidic: Aspartic acid (Asp), Glutamic acid (Glu)(group 4) basic: Asparagine (Asn), Glutamine (Gln), Histidine (His),Lysine (Lys), Arginine (Arg)(group 5) residues that influence chain orientation: Glycine (Gly),Proline (Pro); and(group 6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine(Phe)

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another.

TABLE 1A Amino acid substitution Original Exemplary Conservative residuesubstitution substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln, His, Lys, Arg, Asp Gln Asp (D) Glu, Asn Glu Cys (C)Ser, Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp, Gln Asp Gly (G) Ala AlaHis (H) Asn, Gln, Lys, Arg, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leunorleucine Leu (L) Norleucine, Ile, Val, Met, Ile Ala, Phe Lys (K) Arg,Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, TyrTyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe TyrTyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, LeuNorleucine

Variation in the amino acid sequence of the variant antibody or antigenbinding fragment may include an amino acid addition, deletion,insertion, substitution etc., one or more modification in the backboneor side-chain of one or more amino acid, or an addition of a group oranother molecule to one or more amino acids (side-chains or backbone).

Variant antibody or antigen binding fragment may have substantialsequence similarity and/or sequence identity in its amino acid sequencein comparison with that the original antibody or antigen bindingfragment amino acid sequence. The degree of similarity between twosequences is based upon the percentage of identities (identical aminoacids) and of conservative substitution.

Generally, the degree of similarity and identity between variable chainshas been determined herein using the Blast2 sequence program (Tatiana A.Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool forcomparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250) using default settings, i.e., blastp program, BLOSUM62matrix (open gap 11 and extension gap penalty 1; gapx dropoff 50, expect10.0, word size 3) and activated filters.

Percent identity will therefore be indicative of amino acids which areidentical in comparison with the original peptide and which may occupythe same or similar position. Percent similarity will be indicative ofamino acids that are identical and those that are replaced withconservative amino acid substitution in comparison with the originalpeptide at the same or similar position.

Variants of the present invention therefore comprise those which mayhave at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity with an original sequence or a portion of an original sequence.

Exemplary embodiments of variants are those having at least 81% sequenceidentity to a sequence described herein and 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% sequence similarity with an original sequence or a portion of anoriginal sequence.

Other exemplary embodiments of variants are those having at least 82%sequence identity to a sequence described herein and 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% sequence similarity with an original sequence or a portion of anoriginal sequence.

Further exemplary embodiments of variants are those having at least 85%sequence identity to a sequence described herein and 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequencesimilarity with an original sequence or a portion of an originalsequence.

Other exemplary embodiments of variants are those having at least 90%sequence identity to a sequence described herein and 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with anoriginal sequence or a portion of an original sequence.

Additional exemplary embodiments of variants are those having at least95% sequence identity to a sequence described herein and 95%, 96%, 97%,98%, 99% or 100% sequence similarity with an original sequence or aportion of an original sequence.

Yet additional exemplary embodiments of variants are those having atleast 97% sequence identity to a sequence described herein and 97%, 98%,99% or 100% sequence similarity with an original sequence or a portionof an original sequence.

For a purpose of concision the applicant provides herein a Table 1Billustrating exemplary embodiments of individual variants encompassed bythe present invention and comprising the specified % sequence identityand % sequence similarity. Each “X” is to be construed as defining agiven variant.

TABLE 1B Percent (%) sequence identity 80 81 82 83 84 85 86 87 88 89 9091 92 93 94 95 96 97 98 99 100 Percent 80 X (%) 81 X X 82 X X X 83 X X XX 84 X X X X X 85 X X X X X X 86 X X X X X X X 87 X X X X X X X X 88 X XX X X X X X X 89 X X X X X X X X X X 90 X X X X X X X X X X X 91 X X X XX X X X X X X X 92 X X X X X X X X X X X X X 93 X X X X X X X X X X X XX X 94 X X X X X X X X X X X X X X X 95 X X X X X X X X X X X X X X X X96 X X X X X X X X X X X X X X X X X 97 X X X X X X X X X X X X X X X XX X 98 X X X X X X X X X X X X X X X X X X X 99 X X X X X X X X X X X XX X X X X X X X 100 X X X X X X X X X X X X X X X X X X X X X

The present invention encompasses CDRs, light chain variable regions,heavy chain variable regions, light chains, heavy chains, antibodiesand/or antigen binding fragments which comprise at least 70% identity orat least 80% identity with the sequence described herein.

The present invention therefore encompass, antibodies and antigenbinding fragment which are capable of specific binding to KAAG1 andwhich may comprise sequences selected from the group consisting of:

-   -   a. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:16 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:18,    -   b. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:20 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:22;    -   c. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:24 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:26;    -   d. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:48 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:46;    -   e. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:103 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:126,    -   f. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:104 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:127,    -   g. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:105 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:128,    -   h. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:106 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:145,    -   i. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:107 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:128,    -   j. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:108 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:130,    -   k. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:109 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:141,    -   l. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:110 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:131,    -   m. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:111 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:134,    -   n. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:112 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:135,    -   o. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:113 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:136,    -   p. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:114 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:133,    -   q. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:115 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:140,    -   r. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:116 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:137,    -   s. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:117 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:144,    -   t. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:118 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:139,    -   u. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:119 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:132,    -   v. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:120 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:142,    -   w. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:121 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:138,    -   x. the light chain variable region having at least 70% sequence        identity with SEQ ID NO.:122 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:146,    -   y. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:123 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:147, or;    -   z. a light chain variable region having at least 70% sequence        identity with SEQ ID NO.:124 and a heavy chain variable region        having at least 70% sequence identity with SEQ ID NO.:143.

In accordance with the present invention, the variant antibodies orantigen binding fragments may comprise CDRs that are identical to thoseof the corresponding light chain and/or heavy chain variable region. Inother instance the variant antibodies or antigen binding fragments maycomprise variant CDR(s).

Therefore, exemplary embodiments of a variant antibody or antigenbinding fragment of the present invention are those comprising a lightchain variable region comprising a sequence which is at least 70%, 75%,80% identical to SEQ ID NOs.:16, 20, 24, 103, 106 or 121. The CDRs ofsuch variant may be identical to those of the corresponding non-variant(wild type sequence) antibody or antigen binding fragment or may vary by1-3 amino acids.

Another exemplary embodiment of a variant antibody light chain variableregion encompasses a light chain variable region having CDR amino acidsequences that are 100% identical to the CDR amino acid sequence of SEQID NO.:16 and having for example from 1 to 22 amino acid modifications(e.g., conservative or non-conservative amino acid substitutions) in itsframework region in comparison with the framework region of SEQ IDNO.:16. A SEQ ID NO.:16 variant is provided in SEQ ID NO.:168.

An exemplary embodiment of a variant antibody light chain variableregion encompasses a light chain variable region having CDR amino acidsequences that are 100% identical to the CDR amino acid sequence of SEQID NO.:20 and having for example from 1 to 22 amino acid modifications(e.g., conservative or non-conservative amino acid substitutions) in itsframework region in comparison with the framework region of SEQ IDNO.:20.

An exemplary embodiment of a variant antibody light chain variableregion encompasses a light chain variable region having CDR amino acidsequences that are 100% identical to the CDR amino acid sequence of SEQID NO.:24 and having for example from 1 to 21 amino acid modifications(e.g., conservative or non-conservative amino acid substitutions) in itsframework region in comparison with the framework region of SEQ IDNO.:24. A SEQ ID NO.:24 variant is provided in SEQ ID NO.:172.

An exemplary embodiment of a variant antibody light chain variableregion encompasses a light chain variable region having CDR amino acidsequences that are 100% identical to the CDR amino acid sequence of SEQID NO.:103 and having for example from 1 to 22 amino acid modifications(e.g., conservative or non-conservative amino acid substitutions) in itsframework region in comparison with the framework region of SEQ IDNO.:103.

An exemplary embodiment of a variant antibody light chain variableregion encompasses a light chain variable region having CDR amino acidsequences that are 100% identical to the CDR amino acid sequence of SEQID NO.:106 and having for example from 1 to 22 amino acid modifications(e.g., conservative or non-conservative amino acid substitutions) in itsframework region in comparison with the framework region of SEQ IDNO.:106.

An exemplary embodiment of a variant antibody light chain variableregion encompasses a light chain variable region having CDR amino acidsequences that are 100% identical to the CDR amino acid sequence of SEQID NO.:121 and having for example from 1 to 21 amino acid modifications(e.g., conservative or non-conservative amino acid substitutions) in itsframework region in comparison with the framework region of SEQ IDNO.:121.

In some instances, the variant antibody light chain variable region maycomprise amino acid deletions or additions (in combination or not withamino acid substitutions). Often 1, 2, 3, 4 or 5 amino acid deletions oradditions may be tolerated.

Other exemplary embodiments of a variant antibody or antigen bindingfragment of the present invention are those comprising a heavy chainvariable region comprising a sequence which is at least 70%, 75%, 80%identical to 18, 22, 26, 126, 138 or 145. The CDRs of such variant maybe identical to those of the corresponding non-variant (wild typesequence) antibody or antigen binding fragment or may vary by 1-3 aminoacids.

An exemplary embodiment of a variant antibody heavy chain variableregion encompasses a heavy chain variable region having CDR amino acidsequences that are 100% identical to the CDR amino acid sequence of SEQID NO.:18 and having, for example, from 1 to 22 amino acid modifications(e.g., conservative or non-conservative amino acid substitutions) in itsframework region in comparison with the framework region of SEQ IDNO.:18. A SEQ ID NO.:18 variant is provided in SEQ ID NO.:169.

An exemplary embodiment of a variant antibody heavy chain variableregion encompasses a heavy chain variable region having CDR amino acidsequences that are 100% identical to the CDR amino acid sequence of SEQID NO.:22 and having, for example, from 1 to 23 amino acid modifications(e.g., conservative or non-conservative amino acid substitutions) in itsframework region in comparison with the framework region of SEQ IDNO.:22.

An exemplary embodiment of a variant antibody heavy chain variableregion encompasses a heavy chain variable region having CDR amino acidsequences that are 100% identical to the CDR amino acid sequence of SEQID NO.:26 and having, for example, from 1 to 23 amino acid modifications(e.g., conservative or non-conservative amino acid substitutions) in itsframework region in comparison with the framework region of SEQ IDNO.:26. A SEQ ID NO.:26 variant is provided in SEQ ID NO.:173.

An exemplary embodiment of a variant antibody heavy chain variableregion encompasses a heavy chain variable region having CDR amino acidsequences that are 100% identical to the CDR amino acid sequence of SEQID NO.:126 and having, for example, from 1 to 23 amino acidmodifications (e.g., conservative or non-conservative amino acidsubstitutions) in its framework region in comparison with the frameworkregion of SEQ ID NO.:126.

An exemplary embodiment of a variant antibody heavy chain variableregion encompasses a heavy chain variable region having CDR amino acidsequences that are 100% identical to the CDR amino acid sequence of SEQID NO.:145 and having, for example, from 1 to 23 amino acidmodifications (e.g., conservative or non-conservative amino acidsubstitutions) in its framework region in comparison with the frameworkregion of SEQ ID NO.:145.

An exemplary embodiment of a variant antibody heavy chain variableregion encompasses a heavy chain variable region having CDR amino acidsequences that are 100% identical to the CDR amino acid sequence of SEQID NO.:138 and having, for example, from 1 to 22 amino acidmodifications (e.g., conservative or non-conservative amino acidsubstitutions) in its framework region in comparison with the frameworkregion of SEQ ID NO.:138.

In some instances, the variant antibody heavy chain variable region maycomprise amino acid deletions or additions (in combination or not withamino acid substitutions). Often 1, 2, 3, 4 or 5 amino acid deletions oradditions may be tolerated.

Variant CDRS

Also encompassed by the present invention are polypeptides, antibodiesor antigen binding fragments comprising variable chains having at leastone conservative amino acid substitution in at least one of the CDRsdescribed herein (in comparison with the original CDR).

The present invention also encompasses are polypeptides, antibodies orantigen binding fragments comprising variable chains having at least oneconservative amino acid substitution in at least two of the CDRs (incomparison with the original CDRs).

The present invention also encompasses are polypeptides, antibodies orantigen binding fragments comprising variable chains having at least oneconservative amino acid substitution in the 3 CDRs (in comparison withthe original CDRs).

The present invention also encompasses are polypeptides, antibodies orantigen binding fragments comprising variable chains having at least twoconservative amino acid substitutions in at least one of the CDRs (incomparison with the original CDRs).

The present invention also encompasses are polypeptides, antibodies orantigen binding fragments comprising variable chains having at least twoconservative amino acid substitutions in at least two of the CDRs (incomparison with the original CDRs).

The present invention also encompasses are polypeptides, antibodies orantigen binding fragments comprising variable chains having at least twoconservative amino acid substitutions in the 3 CDRs (in comparison withthe original CDRs).

Comparison of the amino acid sequences of the light chain variableregions or the heavy chain variable regions of antibodies showing thegreatest characteristics allowed us to derive consensus sequences withinthe CDRs and within the variable regions. The consensus for CDRs areprovided in SEQ ID Nos: 72 to 88.

The present invention therefore provides in an exemplary embodiment, anisolated antibody or antigen binding fragment comprising a light chainvariable region having;

-   -   a. a CDRL1 sequence selected from the group consisting of SEQ ID        NO.:72 and SEQ ID NO.:73;    -   b. a CDRL2 sequence selected from the group consisting of SEQ ID        NO.:74, SEQ ID NO.: 75 and SEQ ID NO.:76, or;    -   c. a CDRL3 sequence selected from the group consisting of SEQ ID        NO.:77, SEQ ID NO.:78 and SEQ ID NO.:79.

The present invention therefore provides in an exemplary embodiment, anisolated antibody or antigen binding fragment comprising a heavy chainvariable region having;

-   -   a. a CDRH1 sequence comprising SEQ ID NO.:80;    -   b. a CDRH2 sequence selected from the group consisting of SEQ ID        NO.:81, SEQ ID NO.:82, SEQ ID NO.:83, SEQ ID NO.:84 and SEQ ID        NO.:85, or;    -   c. a CDRH3 sequence selected from the group consisting of SEQ ID        NO.:86, SEQ ID NO.:87 and SEQ ID NO.:88.

In accordance with the present invention, the antibody may comprise aCDRL1 sequence comprising or consisting of formula:

(SEQ ID NO.: 72)X_(1a)SSX2_(a)SLLX_(3a)X_(4a)X_(5a)X_(6a)X_(7a)X_(8a)X_(9a)X_(10a)LX_(11a)

wherein X_(1a) may be a basic amino acid;

wherein X_(2a) may be a basic amino acid;

wherein X_(3a) may be H, Y or N;

wherein X_(4a) may be S, T, N or R;

wherein X_(5a) may be absent, S or N;

wherein X_(6a) may be D, F or N;

wherein X_(7a) may be G or Q;

wherein X_(8a) may be K, L or N;

wherein X_(9a) may be T or N;

wherein X_(10a) may be an aromatic amino acid, and;

wherein X_(11a) may be A, N, E or Y.

In an exemplary embodiment of the invention X_(1a) may be K or R.

In a further embodiment of the invention X_(2a) may be Q or K.

In yet a further embodiment of the invention X₃, may be N or H.

In an additional embodiment of the invention X_(10a) may be Y or F.

More specific embodiments of the invention include CDRL1 of SEQ IDNO.:72 where: X_(1a) is K; X_(2a) is Q; X_(3a) is N; X_(3a) is H; X_(4a)is S; X_(4a) is T; X_(5a) is S; X_(5a) is absent; X_(6a) is N; X_(7a) isQ; X_(7a) is G; X_(8a) is K; X_(9a) is N; X_(9a) is T; X_(10a) is Y; orX_(11a) is A.

In accordance with the present invention, the antibody may comprise aCDRL1 sequence comprising or consisting of formula:

(SEQ ID NO.: 73) KASQDX_(1b)X_(2b)X_(3b)X_(4b)X5_(b)X_(6b)

wherein X_(1b) may be an hydrophobic amino acid;

wherein X_(2b) may be G or H;

wherein X_(3b) may be T, N or R;

wherein X_(4b) may be F, Y or A;

wherein X_(5b) may be an hydrophobic amino acid, and;

wherein X_(6b) may be N or A.

In an exemplary embodiment of the invention X_(1b) may be V or I.

In another exemplary embodiment of the invention X_(5b) may be V or L.

More specific embodiments of the invention include CDRL1 of SEQ IDNO.:73 where X_(1b) is I; X_(2b) is H; X_(3b) is T; X_(3b) is N; X_(4b)is Y; X_(4b) iS F; X_(5b) is L or X_(6b) is N.

Other exemplary embodiments of CDRL1 are provided in SEQ ID NOs. 89 and90.

In accordance with the present invention, the antibody may comprise aCDRL2 sequence comprising or consisting of formula:

(SEQ ID NO.: 74) FX_(1c)STX_(2c)X_(3c)S

Wherein X_(1c) is A or G;

Wherein X_(2c) is R or T, and;

Wherein X_(3c) is E, K or A.

In an exemplary embodiment of the invention X_(1c) may be A and X_(2c)may be T.

In another exemplary embodiment of the invention X_(1c) may be A andX_(2c) may be R.

Other specific embodiments of the invention include CDRL2 of SEQ IDNO.:74 where X_(1c) is A; X_(2c) is R or X_(3c) is E.

In accordance with the present invention, the antibody may comprise aCDRL2 sequence comprising or consisting of formula:

(SEQ ID NO.: 75) X_(1d)VSX_(2d)X_(3d)X_(4d)S

Wherein X_(1d) may be L or K;

Wherein X_(2d) may be a basic amino acid;

Wherein X_(3d) may be L or R and;

Wherein X_(4d) may be D or F.

In an exemplary embodiment of the invention X_(2d) may be K or N.

Other specific embodiments of the invention include CDRL2 of SEQ IDNO.:75 where X_(1d) is L; X_(2d) is K; X_(3d) is L or X_(4d) is D.

In accordance with the present invention, the antibody may comprise aCDRL2 sequence comprising or consisting of formula:

(SEQ ID NO.: 76) X_(1e)ANRLVX_(2e)

Wherein X_(1e) may be a basic amino acid, and;

Wherein X_(2e) may be D or A.

In an exemplary embodiment of the invention X_(1e) may be R or H.

Other specific embodiments of the invention include CDRL2 of SEQ IDNO.:76 where X_(1e) is R or X_(2e) is D.

Other exemplary embodiments of CDRL2 are provided in SEQ ID NOs.: 91-93.

In accordance with the present invention, the antibody may comprise aCDRL3 sequence comprising or consisting of formula:

(SEQ ID NO.: 77) X_(1f)QX_(2f)X_(3f)X_(4f)X_(5f)PLT 

Wherein X_(1f) may be Q or L;

Wherein X_(2f) may be an aromatic amino acid;

Wherein X_(3f) may be D, F or Y;

Wherein X_(4f) may be E, A, N or S, and;

Wherein X_(5f) may be I, F or T.

In an exemplary embodiment of the invention X_(2f) may be Y or H.

In another exemplary embodiment of the invention X_(3f) may be Y or D.

In yet another exemplary embodiment of the invention X_(5f) may be I orT.

Other specific embodiments of the invention include CDRL3 of SEQ IDNO.:77 where X_(1f) is Q; X_(2f) iS H; X_(3f) is D; X_(3f) is Y; X_(4f)is S; X_(4f) is E; X_(4f) is A; X_(5f) is T, or X_(5f) is I.

In accordance with the present invention, the antibody may comprise aCDRL3 sequence comprising or consisting of formula:

(SEQ ID NO.: 78) QQHX_(1g)X_(2g)X_(3g)PLT 

Wherein X_(1g) may be an aromatic amino acid;

Wherein X_(2g) may be N or S, and;

Wherein X_(3g) may be I or T.

In an exemplary embodiment of the invention X_(1g) may be F or Y

Other specific embodiments of the invention include CDRL3 of SEQ IDNO.:78 where X_(2g) is S or X_(3g) is T.

In accordance with the present invention, the antibody may comprise aCDRL3 sequence comprising or consisting of formula:

(SEQ ID NO.: 79) X_(1h)QGX_(2h)HX_(3h)PX_(4h)T

Wherein X_(1h) may be an aromatic amino acid;

Wherein X_(2h) may be a neutral hydrophilic amino acid;

Wherein X_(3h) may be F or V, and;

Wherein X_(4h) may be R or L.

In an exemplary embodiment of the invention X_(1h) may be W or F.

In another exemplary embodiment of the invention X_(2h) may be S or T.

Other specific embodiments of the invention include CDRL3 of SEQ IDNO.:79 where X_(1h) is W; X_(2h) is T; X_(3h) is F, or X_(4h) is R.

Other exemplary embodiments of CDRL3 are provided in SEQ ID NOs. 94 and95.

In accordance with the present invention, the antibody may comprise aCDRH1 sequence comprising or consisting of formula:

(SEQ ID NO.: 80) GYX_(1i)FX_(2i)X_(3i)YX_(4i)X_(5i)H

Wherein X_(1i) may be T, I or K;

Wherein X_(2i) may be a neutral hydrophilic amino acid;

Wherein X_(3i) may be an acidic amino acid;

Wherein X_(4i) may be E, N or D, and;

Wherein X_(5i) may be hydrophobic amino acid.

In an exemplary embodiment of the invention X_(2i) may be T or S.

In another exemplary embodiment of the invention X_(3i) may be D or E.

In yet another exemplary embodiment of the invention X_(4i) may be N orE.

In a further exemplary embodiment of the invention X_(5i) may be M, I orv.

Other specific embodiments of the invention include CDRH1 of SEQ IDNO.:80 where X_(2i) is T; X_(3i) is D; X_(4i) is E; X_(5i) is I orX_(5i) is M.

Other exemplary embodiments of CDRH1 are provided in SEQ ID NOs.: 96 and97.

In accordance with the present invention, the antibody may comprise aCDRH2 sequence comprising or consisting of formula:

(SEQ ID NO.: 81) X_(1j)X_(2j)DPX_(3j)TGX_(4j)TX_(5j) 

Wherein X_(1j) may be V or G

Wherein X_(2j) may be a hydrophobic amino acid;

Wherein X_(3j) may be A, G or E;

Wherein X_(4j) may be R, G, D, A, S, N or V, and;

Wherein X_(5j) may be a hydrophobic amino acid.

In an exemplary embodiment of the invention X_(2j) may be I or L.

In another exemplary embodiment of the invention X_(5j) may be A or V.

Other specific embodiments of the invention include CDRH2 of SEQ IDNO.:81 where X_(1j) is V; X_(2j) is I; X_(3j) is E; X_(4j) is D orX_(5j) is A.

In accordance with the present invention, the antibody may comprise aCDRH2 sequence comprising or consisting of formula:

(SEQ ID NO.: 82) VX_(1k)DPX_(2k)TGX_(3k)TA

Wherein X_(1k) may be an hydrophobic amino acid;

Wherein X_(2k) may be A, E or G;

Wherein X_(3k) may be R, G, A, S, N V or D.

In an exemplary embodiment of the invention X_(1k) may be L or I.

Other specific embodiments of the invention include CDRH2 of SEQ IDNO.:82 where X_(1k) is I; X_(2k) is E, or X_(3k) is D.

In accordance with the present invention, the antibody may comprise aCDRH2 sequence comprising or consisting of formula:

(SEQ ID NO.: 83) YIX_(1l)X_(2l)X_(3l)GX_(4l)X_(5l)X_(6l)

Wherein X1_(l) may be S or N;

Wherein X_(2l) may be an aromatic amino acid

Wherein X_(3l) may be D, E or N;

Wherein X4_(l) may be a D or H;

Wherein X_(5l) may be Y, S or N;

Wherein X_(6l) may be D, E or N.

In an exemplary embodiment of the invention X_(3l) may be D or N.

In another exemplary embodiment of the invention X_(6l) may be D or N.

Other specific embodiments of the invention include CDRH2 of SEQ IDNO.:83 where X_(2l) is F or Y, X_(3l) is N, X_(4l) is D or X_(6l) is N.

In accordance with the present invention, the antibody may comprise aCDRH2 sequence comprising or consisting of formula:

X_(1m)INPYNX_(2m)VTE  (SEQ ID NO.:84)

wherein X_(1m) may be N or Y, and;

wherein X_(2m) may be E, D or N.

In an exemplary embodiment of the invention X_(2m) may be D or N.

Other specific embodiments of the invention include CDRH2 of SEQ IDNO.:84 where X_(1m) is N or X_(2m) is D.

In accordance with the present invention, the antibody may comprise aCDRH2 sequence comprising or consisting of formula:

(SEQ ID NO.: 85) DINPX_(1n)YGX_(2n)X_(3n)T 

Wherein X_(1n) may be N or Y,

Wherein X_(2n) may be G or T and;

wherein X_(3n) may be I or T.

Other exemplary embodiments of CDRH2 are provided in SEQ ID NOs. 98 and99.

In accordance with the present invention, the antibody may comprise aCDRH3 sequence comprising or consisting of formula:

(SEQ ID NO.: 86) MX_(1o)X_(2o)X_(3o)DY

Wherein X_(1o) may be G or S;

Wherein X_(2o) may be Y or H, and;

wherein X_(3o) may be A or S.

Other specific embodiments of the invention include CDRH3 of SEQ IDNO.:86 where X_(1o) is G; X_(2o) is Y or X_(3o) is S.

In accordance with the present invention, the antibody may comprise aCDRH3 sequence comprising or consisting of formula:

(SEQ ID NO.: 87) IX_(1p)YAX_(2p)DY 

Wherein X_(1p) may be G or S and;

Wherein X_(2p) may be absent or M.

Other specific embodiments of the invention include CDRH3 of SEQ IDNO.:87 where X_(1p) is S or X_(2p) is M.

In accordance with the present invention, the antibody may comprise aCDRH3 sequence comprising or consisting of formula:

(SEQ ID NO.: 88) AX_(1q)X_(2q)GLRX_(3q)

Wherein X_(1q) may be R or W;

Wherein X_(2q) may be an aromatic amino acid and;

wherein X_(3q) may be a basic amino acid.

In an exemplary embodiment of the invention X_(2q) may be W or F.

In another exemplary embodiment of the invention X_(3q) may be Q or N.

Other specific embodiments of the invention include CDRH3 of SEQ IDNO.:88 where X_(1q) is R; X_(2q) is W or X_(3q) is N.

Variant antibodies or antigen binding fragments encompassed by thepresent invention include those that may comprise an insertion, adeletion or an amino acid substitution (conservative ornon-conservative). These variants may have at least one amino acidresidue in its amino acid sequence removed and a different residueinserted in its place.

Humanized Antibodies

Exemplary embodiments of variant antibodies and antigen bindingfragments of the present invention are a group of antibodies and antigenbinding fragments capable of binding to KAAG1 and characterized hereinas being humanized.

The humanized antibodies and antigen binding fragments of the presentinvention includes more particularly, humanized 3D3, 3A4 or 3C4antibodies and antigen binding fragments. The humanized 3D3, 3A4 or 3C4antibodies have at least one amino acid difference in a framework regionin comparison with the monoclonal 3D3, 3A4 or 3C4 antibody.

Humanized 3A4 antibodies having CDRs identical to those of themonoclonal 3A4 antibody (VL: SEQ ID NO.:48, VH: SEQ ID NO.:46) weregenerated and tested. These humanized antibodies comprise up to 11 aminoacid substitutions (from one to eleven) in the variable light chainframework region and up to 23 amino acid substitutions (from one totwenty-three) in the variable heavy chain framework region in comparisonwith the monoclonal 3A4 antibody. The applicant has shown that thesehumanized 3A4 antibodies bind to KAAG1 as efficiently as the monoclonal3A4 antibody.

Exemplary embodiments of variant antibody or antigen binding fragmentsinclude those having a light chain variable region as set forth in SEQID NO.:186:

SEQ ID NO.: 186 DXVMTQTPLSLXVXXGXXASISCRSSQSLLHSNGNTYLEWYLQKPGQSPXLLIHTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDXGVYYCFQGSHVP LTFGXGTXLEXK,wherein at least one of the amino acids identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:48.The amino acid substitution may be, for example, an amino acid found ata corresponding position of a natural human antibody or a human antibodyconsensus. The amino acid substitution may be, for example conservative.

Another exemplary embodiment of a variant antibody or antigen bindingfragment include those having a light chain variable region as set forthin SEQ ID NO.:187:

SEQ ID NO.: 187DX_(e1)VMTQTPLSLX_(e2)VX_(e3)X_(e4)GX_(e5)X_(e6)ASISCRSSQSLLHSNGNTYLEWYLQKPGQSPX_(e7)LLIHTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDX_(e8)GVYYCFQGSHVPLTFGX_(e9)GTX_(e10)LEX_(e11)K,Wherein X_(e1) may be a hydrophobic amino acid;

Wherein X_(e2) may be A or P;

Wherein X_(e3) may be neutral hydrophilic amino acid;

Wherein X_(e4) may be L or P;

Wherein X_(e5) may be an acidic amino acid;

Wherein X_(e6) may be Q or P;

Wherein X_(e7) may be a basic amino acid;Wherein X_(e8) may be a hydrophobic amino acid;

Wherein X_(e9) may be A or Q;

Wherein X_(e10) may be a basic amino acid; orWherein X_(e11) may be a hydrophobic amino acid,wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:48.

An additional exemplary embodiment of a variant antibody or antigenbinding fragment include those having a light chain variable region asset forth in SEQ ID NO.:188:

SEQ ID NO.: 188DX_(e1)VMTQTPLSLX_(e2)VX_(e3)X_(e4)GX_(e5)X_(e6)ASISCRSSQSLLHSNGNTYLEWYLQKPGQSPX_(e7)LLIHTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDX_(e8)GVYYCFQGSHVPLTFGX_(e9)GTX_(e10)LEX_(e11)K

Wherein X_(E1) may be V or I Wherein X_(E2) may be A or P Wherein X_(E3)may be S or T Wherein X_(E4) may be L or P Wherein X_(E5) may be D or EWherein X_(E6) may be Q or P Wherein X_(E7) may be K or Q Wherein X_(E8)may be L or V Wherein X_(E9) may be A or Q Wherein X_(E10) may be R or Kor Wherein X_(E11) may be L or I,

wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:48.

In accordance with an embodiment, the light chain variable domainvariant may have a sequence as set forth in SEQ ID NO.:189 or 190:

SEQ ID NO.: 189 DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP LTFGQGTKLEIK.SEQ ID NO.: 190 DVVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP LTFGQGTKLEIK.

Exemplary embodiments of variant antibody or antigen binding fragmentsinclude those having a heavy chain variable region as set forth in SEQID NO.:191.

SEQ ID NO.: 191 QXQLVQSGXEXXKPGASVKXSCKASGYTFTDDYMSWVXQXXGXXLEWXGDINPYNGDTNYNQKFKGXXXXTXDXSXSTAYMXLXSLXSEDXAVYYCARDP GAMDYWGQGTXVTVSS,wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:46.The amino acid substitution may be, for example, an amino acid found ata corresponding position of a natural human antibody or a human antibodyconsensus. The amino acid substitution may be, for example conservative.

Another exemplary embodiment of a variant antibody or antigen bindingfragment include those having a heavy chain variable region as set forthin SEQ ID NO.:192:

SEQ ID NO.: 192QX_(f1)QLVQSGX_(f2)EX_(f3)X_(bf4)KPGASVKX_(f5)SCKASGYTFTDDYMSWVX_(f6)QX_(f7)X_(f8)GX_(f9)X_(f10)LEWX_(f11)GDINPYNGDTNYNQKFKGX_(f12)X_(f13)X_(b14)X_(f15)TX_(f16)DX_(f17)SX_(f18)STAYMX_(f19)LX_(f20)SLX_(f21)SEDX_(f22)AVYYCARDPGAMDYWGQGTX_(f23)VTVSS,Wherein X_(f1) may be a hydrophobic amino acid;

Wherein X_(bf2) may be P or A;

Wherein X_(f3) may be a hydrophobic amino acid;

Wherein X_(f4) may be V or K;

Wherein X_(f5) may be a hydrophobic amino acid;Wherein X_(f6) may be a basic amino acid;

Wherein X_(f7) may be S or A; Wherein X_(f8) may be H or P;

Wherein X_(f9) may be a basic amino acid;

Wherein X_(f10) may be S or G;

Wherein X_(f11) may be a hydrophobic amino acid;Wherein X_(f12) may be a basic amino acid;Wherein X_(f13) may be a hydrophobic amino acid;

Wherein X_(f14) may be I or T;

Wherein X_(f15) may be a hydrophobic amino acid;Wherein X_(f16) may be a hydrophobic amino acid;

Wherein X_(f17) may be K or T;

Wherein X_(f18) may be a neutral hydrophilic amino acid;

Wherein X_(f19) may be Q or E; Wherein X_(f20) may be N or S; WhereinX_(f21) may be T or R;

Wherein X_(f22) may be a neutral hydrophilic amino acid; or

Wherein X_(f23) may be S or L,

wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:46.

An additional exemplary embodiment of a variant antibody or antigenbinding fragment include those having a heavy chain variable region asset forth in SEQ ID NO.:193:

SEQ ID NO.: 193QX_(F1)QLVQSGX_(F2)EX_(F3)X_(F4)KPGASVKX_(F5)SCKASGYTFTDDYMSWVX_(F6)QX_(F7)X_(F8)GX_(F9)X_(F10)LEWX_(F11)GDINPYNGDTNYNQKFKGX_(F12)X_(F13)X_(F14)X_(F15)TX_(F16)DX_(F17)SX_(F18)STAYMX_(F19)LX_(F20)SLX_(F21)SEDX_(F22)AVYYCARDPGAMDYWGQGTX_(F23)VTVSS

Wherein X_(F1) may be I or V; Wherein X_(F2) may be P or A; WhereinX_(F3) may be M or V; Wherein X_(F4) may be V or K; Wherein X_(F5) maybe M or V; Wherein X_(F6) may be K or R; Wherein X_(F7) may be S or A;Wherein X_(F8) may be H or P; Wherein X_(F9) may be K or Q; WhereinX_(F10) may be S or G; Wherein X_(F11) may be I or M; Wherein X_(F12)may be K or R; Wherein X_(F13) may be A or V; Wherein X_(F14) may be Ior T; Wherein X_(F15) may be L or I; Wherein X_(F16) may be V or A;Wherein X_(F17) may be K or T; Wherein X_(F18) may be S or T; WhereinX_(F19) may be Q or E; Wherein X_(F20) may be N or S; Wherein X_(F21)may be T or R; Wherein X_(F22) may be S or T; or Wherein X_(F23) is S orL,

wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:46.

In accordance with an embodiment, the heavy chain variable domainvariant may have a sequence as set forth in any one of SEQ ID NO. 194 to197:

SEQ ID NO.: 194 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWMGDINPYNGDTNYNQKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCARDP GAMDYWGQGTLVTVSS.SEQ ID NO.: 195 QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWMGDINPYNGDTNYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDP GAMDYWGQGTLVTVSS.SEQ ID NO.: 196 QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWIGDINPYNGDTNYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARDP GAMDYWGQGTLVTVSS.SEQ ID NO.: 197 QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGDTNYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDP GAMDYWGQGTLVTVSS.

In accordance with an embodiment of the invention, the humanized 3D3antibody may have a light chain variable region of formula:

(SEQ ID NO.: 174) DIVMTQSPXSLAVSXGXXXTXNCKSSQSLLNSNFQKNFLAWYQQKPGQXPKLLIYFASTRESSXPDRFXGSGSGTDFTLTISSXQAEDXAXYXCQQHYST PLTFGXGTKLEXK;

wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:16.The amino acid substitution may be, for example conservative.

In accordance with a more specific embodiment, the humanized 3D3antibody may have a light chain variable region of formula:

(SEQ ID NO.: 175)DIVMTQSPX_(A1)SLAVSX_(A2)GX_(A3)X_(A4)X_(A5)TX_(A6)NCKSSQSLLNSNFQKNFLAWYQQKPGQX_(A7)PKLLIYFASTRESSX_(A8)PDRFX_(A9)GSGSGTDFTLTISSX_(A10)QAEDX_(A11)AX_(A12)YX_(A13)CQQHYSTPLTFGX_(A14)GTKLEX_(A15)K;

-   -   Wherein X_(A1) may be, for example, D or S;    -   Wherein X_(A2) may be, for example, a hydrophobic amino acid or        more particularly L or I;    -   Wherein X_(A3) may be, for example, E or Q;    -   Wherein X_(A4) may be, for example, a basic amino acid or more        particularly R or K;    -   Wherein X_(A5) may be, for example, a hydrophobic amino acid or        more particularly A or V;    -   Wherein X_(A6) may be, for example, a hydrophobic amino acid or        more particularly I or M;    -   Wherein X_(A7) may be, for example, P or S;    -   Wherein X_(A8) may be, for example, a hydrophobic amino acid or        more particularly V or I;    -   Wherein X_(A9) may be, for example, S or I;    -   Wherein X_(A10) may be, for example, a hydrophobic amino acid or        more particularly L or V;    -   Wherein X_(A11) may be, for example, a hydrophobic amino acid or        more particularly V or L;    -   Wherein X_(A12) may be, for example, V or D;    -   Wherein X_(A13) may be, for example, an aromatic amino acid or        more particularly Y or F;    -   Wherein X_(A14) may be, for example, Q or A and;    -   Wherein X_(A15) may be, for example, a hydrophobic amino acid or        more particularly I or L.

In accordance with an even more specific embodiment, the humanized 3D3antibody may have a light chain variable region of formula:

(SEQ ID NO.: 176)DIVMTQSPX_(a1)SLAVSX_(a2)GX_(a3)X_(a4)X_(a5)TX_(a6)NCKSSQSLLNSNFQKNFLAWYQQKPGQX_(a7)PKLLIYFASTRESSX_(a8)PDRFX_(a9)GSGSGTDFTLTISSX_(a10)QAEDX_(a11)AX_(a12)YX_(a13)CQQHYSTPLTFGX_(a14)GTKLEX_(a15)K;

Wherein X_(a1) may be, for example, D or S;

Wherein X_(a2) may be, for example, L or I;

Wherein X_(a3) may be, for example, E or Q;

Wherein X_(a4) may be, for example, R or K;

Wherein X_(a5) may be, for example, A or V;

Wherein X_(a6) may be, for example, I or M;

Wherein X_(a7) may be, for example, P or S;

Wherein X_(a8) may be, for example, V or I;

Wherein X_(a9) may be, for example, S or I;

Wherein X_(a10) may be, for example, L or V;

Wherein X_(a11) may be, for example, V or L;

Wherein X_(a12) may be, for example, V or D;

Wherein X_(a13) may be, for example, Y or F;

Wherein X_(a14) may be, for example, Q or A and;

Wherein X_(a15) is for example, I or L.

In accordance with an embodiment of the present invention, the humanized3D3 antibody may have a heavy chain variable region of formula:

(SEQ ID NO.: 177) EVQLXQSXAEXXXPGASVXXSCKASGYIFTDYEIHWVXQXPXXGLEWXGVIDPETGNTAFNQKFKGXXTXTADXSXSTAYMELSSLTSEDXAVYYCMGYS DYWGQGTXXTVSS;wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:18.The amino acid substitution may be, for example conservative.

In accordance with a more specific embodiment, the humanized 3D3antibody may have a heavy chain variable region of formula:

(SEQ ID NO.: 178)EVQLX_(B1)QSX_(B2)AEX_(B3)XNX_(B5)PGASVX_(B6)X_(B7)SCKASGYIFTDYEIHWVX_(B8)QX_(B9)PX_(B10)X_(B11)GLEWX_(B12)GVIDPETGNTAFNQKFKGX_(B13)X_(B14)TX_(B15)TADX_(B16)SX_(B17)STAYMELSSLTSEDX_(B18)AVYYCMGYSDYWGQGTX_(B19)X_(B20)TV SS,

-   -   Wherein X_(B1) may be, for example, V or Q;    -   Wherein X_(B2) may be, for example, G or V;    -   Wherein X_(B3) may be, for example, a hydrophobic amino acid or        more particularly V or L;    -   Wherein X_(B4) may be, for example, K or V;    -   Wherein X_(B5) may be, for example, a basic amino acid or more        particularly K or R;    -   Wherein X_(B6) may be, for example, K or T;    -   Wherein X_(B7) may be, for example, a hydrophobic amino acid or        more particularly V or L;    -   Wherein X_(B8) may be, for example, a basic amino acid or more        particularly R or K;    -   Wherein X_(B9) may be, for example, A or T;    -   Wherein X_(B10) may be, for example, G or V;    -   Wherein X_(B11) may be, for example, Q or H;    -   Wherein X_(B12) may be, for example, a hydrophobic amino acid or        more particularly M or I;    -   Wherein X_(B13) may be, for example, a basic amino acid or more        particularly R or K;    -   Wherein X_(B14) may be, for example, a hydrophobic amino acid or        more particularly V or A;    -   Wherein X_(B15) may be, for example, a hydrophobic amino acid or        more particularly I or L;    -   Wherein X_(B16) may be, for example, T or I;    -   Wherein X_(B17) may be, for example, a neutral hydrophilic amino        acid or more particularly T or S;    -   Wherein X_(B18) may be, for example, a neutral hydrophilic amino        acid or more particularly T or S;    -   Wherein X_(B19) may be, for example, L or T and;    -   Wherein X_(B20) may be, for example, a hydrophobic amino acid or        more particularly V or L.

In accordance with a more specific embodiment, the humanized 3D3antibody may have a heavy chain variable region of formula:

(SEQ ID NO.: 179)EVQLX_(b1)QSX_(b2)AEX_(b3)X_(b4)X_(b5)PGASVX_(b6)X_(b7)SCKASGYIFTDYEIHWVX_(b8)QX_(b9)PX_(b10)X_(b11)GLEWX_(b12)GVIDPETGNTAFNQKFKGX_(b13)X_(b14)TX_(b15)TADX_(b16)SX_(b17)STAYMELSSLTSEDX_(b18)AVYYCMGYSDYWGQGTX_(b19)X_(b20)TVSS;

Wherein X_(b1) may be, for example, V or Q;

Wherein X_(b2) may be, for example, G or V;

Wherein X_(b3) may be, for example, V or L;

Wherein X_(b4) may be, for example, K or V;

Wherein X_(b5) may be, for example, K or R;

Wherein X_(b6) may be, for example, K or T;

Wherein X_(b7) may be, for example, V or L;

Wherein X_(b8) may be, for example, R or K;

Wherein X_(b9) may be, for example, A or T;

Wherein X_(b10) may be, for example, G or V;

Wherein X_(b11) may be, for example, Q or H;

Wherein X_(b12) may be, for example, M or I;

Wherein X_(b13) may be, for example, R or K;

Wherein X_(b14) may be, for example, V or A;

Wherein X_(b15) may be, for example, I or L;

Wherein X_(b16) may be, for example, T or I;

Wherein X_(b17) may be, for example, T or S;

Wherein X_(b18) may be, for example, T or S;

Wherein X_(b19) may be, for example, L or T;

Wherein X_(b20) may be, for example, V or L.

In accordance with an embodiment of the present invention, the humanized3C4 antibody may have a light chain variable region of formula:

(SEQ ID NO.: 180) DIVMXQSPSSXXASXGXRVTITCKASQDIHNFLNWFQQKPGKXPKTLIFRANRLVDGVPSRFSGSGSGXDYXLTISSLXXEDXXXYSCLQYDEIPLTFGX GTKLEXX;wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:24.The amino acid substitution may be, for example conservative.

In accordance with a more specific embodiment, the humanized 3C4antibody may have a light chain variable region of formula:

(SEQ ID NO.: 181)DIVMX_(C1)QSPSSX_(C2)X_(C3)ASX_(C4)GX_(C5)RVTITCKASQDIHNFLNWFQQKPGKX_(C6)PKTLIFRANRLVDGVPSRFSGSGSGX_(C7)DYX_(C8)LTISSLX_(C9)X_(C10)EDX_(C11)X_(C12)X_(C13)YSCLQYDEIPLTFGX_(C14)GTKLEX_(C15)X_(C16);

-   -   Wherein X_(C1) may be, for example, a neutral hydrophilic amino        acid or more particularly T or S;    -   Wherein X_(C2) may be, for example, a hydrophobic amino acid or        more particularly L or M;    -   Wherein X_(C3) may be, for example, S or Y;    -   Wherein X_(C4) may be, for example, a hydrophobic amino acid or        more particularly V or L;    -   Wherein X_(C5) may be, for example, an acidic amino acid or more        particularly D or E;    -   Wherein X_(C6) may be, for example, A or S;    -   Wherein X_(C7) may be, for example, T or Q;    -   Wherein X_(C8) may be, for example, a neutral hydrophilic amino        acid or more particularly T or S;    -   Wherein X_(C9) may be, for example, Q or E;    -   Wherein X_(C10) may be, for example, P or F;    -   Wherein X_(C11) may be, for example, F or L;    -   Wherein X_(C12) may be, for example, A or G;    -   Wherein X_(C13) may be, for example, T or I;    -   Wherein X_(C14) may be, for example, Q or A;    -   Wherein X_(C15) may be, for example, a hydrophobic amino acid or        more particularly I or L, and; wherein X_(C16) may be, for        example, a basic amino acid or more particularly K or R.

In accordance with a more specific embodiment, the humanized 3C4antibody may have a light chain variable region of formula:

(SEQ ID NO.: 182)DIVMX_(c1)QSPSSX_(c2)X_(c3)ASX_(c4)GX_(c5)RVTITCKASQDIHNFLNWFQQKPGKX_(c6)PKTLIFRANRLVDGVPSRFSGSGSGX_(c7)DYX_(c8)LTISSLX_(c9)X_(c10)EDX_(c11)X_(c12)X_(c13)YSCLQYDEIPLTFGX_(c14)GTKLEX_(c15)X_(c16);

Wherein X_(c1) may be, for example, T or S;

Wherein X_(c2) may be, for example, L or M;

Wherein X_(c3) may be, for example, S or Y;

Wherein X_(c4) may be, for example, V or L;

Wherein X_(c5) may be, for example, D or E;

Wherein X_(c6) may be, for example, A or S;

Wherein X_(c7) may be, for example, T or Q;

Wherein X_(c8) may be, for example, T or S;

Wherein X_(c9) may be, for example, Q or E;

Wherein X_(c10) may be, for example, P or F;

Wherein X_(c11) may be, for example, F or L;

Wherein X_(c12) may be, for example, A or G;

Wherein X_(c13) may be, for example, T or I;

Wherein X_(c14) may be, for example, Q or A;

Wherein X_(c15) may be, for example, I or L and;

wherein X_(c16) may be, for example, K or R.

In accordance with an embodiment of the present invention, the humanized3C4 antibody may have a heavy chain variable region of formula:

(SEQ ID NO.: 183) EVQLQESGPXLVKPSQXLSLTCTVXGFSITSGYGWHWIRQXPGXXLEWXGYINYDGHNDYNPSLKSRXXIXQDTSKNQFXLXLXSVTXXDTAXYYCAS SYDGLFAYWGQGTLVTVSX;wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:26.The amino acid substitution may be, for example conservative.

In accordance with a more specific embodiment, the humanized 3C4antibody may have a heavy chain variable region of formula:

(SEQ ID NO.: 184) EVQLQESGPX_(D1)LVKPSQX_(D2)LSLTCTVX_(D3)GFSITSGYGWHWIRQX_(D4)PGX_(D5)X_(D6)LEWX_(D7)GYINYDGHNDYNPSLKSRX_(D8)X_(D9)IX_(D10)QDTSKNQFX_(D11)LX_(D12)LX_(D13)SVTX_(D14)X_(D15)DTAX_(D16)YYCASSYDGLFAYWGQGTLVTVSX_(D17);

-   -   Wherein X_(D1) may be, for example, G or D;    -   Wherein X_(D2) may be, for example, a neutral hydrophilic amino        acid or more particularly T or S;    -   Wherein X_(D3) may be, for example, a neutral hydrophilic amino        acid or more particularly S or T;    -   Wherein X_(D4) may be, for example, H or F;    -   Wherein X_(D5) may be, for example, K or N;    -   Wherein X_(D6) may be, for example, G or K;    -   Wherein X_(D7) may be, for example, a hydrophobic amino acid or        more particularly I or M;    -   Wherein X_(D8) may be, for example, a hydrophobic amino acid or        more particularly V or I;    -   Wherein X_(D9) may be, for example, a neutral hydrophilic amino        acid or more particularly T or S;    -   Wherein X_(D10) may be, for example, a neutral hydrophilic amino        acid or more particularly S or T;    -   Wherein X_(D11) may be, for example, a neutral hydrophilic amino        acid or more particularly S or F;    -   Wherein X_(D12) may be, for example, a basic amino acid or more        particularly K or Q;    -   Wherein X_(D13) may be, for example, S or N;    -   Wherein X_(D14) may be, for example, A or T;    -   Wherein X_(D15) may be, for example, A or E;    -   Wherein X_(D16) may be, for example, V or T and;    -   Wherein X_(D17) may be any amino acid, A or absent.

In accordance with a more specific embodiment, the humanized 3C4antibody may have a heavy chain variable region of formula:

(SEQ ID NO.: 185) EVQLQESGPX_(d1)LVKPSQX_(d2)LSLTCTVX_(d3)GFSITSGYGWHWIRQX_(d4)PGX_(d5)X_(d6)LEWX_(d7)GYINYDGHNDYNPSLKSRX_(d8)X_(d9)IX_(d10)QDTSKNQFX_(d11)LX_(d12)LX_(d13)SVTX_(d14)X_(d15)DTAX_(d16)YYCASSYDGLFAYWGQGTLVTVSX_(d17);

Wherein X_(d1) may be, for example, G or D;

Wherein X_(d2) may be, for example, T or S;

Wherein X_(d3) may be, for example, S or T;

Wherein X_(d4) may be, for example, H or F;

Wherein X_(d5) may be, for example, K or N;

Wherein X_(d6) may be, for example, G or K;

Wherein X_(d7) may be, for example, I or M;

Wherein X_(d8) may be, for example, V or I;

Wherein X_(d9) may be, for example, T or S;

Wherein X_(d10) may be, for example, S or T;

Wherein X_(d11) may be, for example, S or F;

Wherein X_(d12) may be, for example, K or Q;

Wherein X_(d13) may be, for example, S or N;

Wherein X_(d14) may be, for example, A or T;

Wherein X_(d15) may be, for example, A or E;

Wherein X_(d16) may be, for example, V or T and;

Wherein X_(d17), A or absent.

Accordingly, the present invention provides in one aspect, an antibodyor antigen binding fragment thereof capable of specific binding toKidney associated antigen 1 (KAAG1) which may have a light chainvariable region at least 70% identical to SEQ ID NO.:16 and/or a heavychain variable region at least 70% identical to SEQ ID NO.:18. Theantibody or antigen binding fragment thereof may also comprise at leastone amino acid substitution in comparison with SEQ ID NO.:16 or SEQ IDNO.:18.

The present invention also provides in another aspect, an antibody orantigen binding fragment thereof which may have a light chain variableregion at least 70% identical to SEQ ID NO.:24 and/or a heavy chainvariable region at least 70% identical to SEQ ID NO.:26. The antibody orantigen binding fragment thereof may also comprise at least one aminoacid substitution in comparison with SEQ ID NO.:24 or SEQ ID NO.:26.

The present invention also provides in another aspect, an antibody orantigen binding fragment thereof which may have a light chain variableregion at least 70% identical to SEQ ID NO.:48 and/or a heavy chainvariable region at least 70% identical to SEQ ID NO.:46. The antibody orantigen binding fragment thereof may also comprise at least one aminoacid substitution in comparison with SEQ ID NO.:48 or SEQ ID NO.:46.

In accordance with an embodiment of the invention, the amino acidsubstitution may be outside of a complementarity determining region(CDR). An antibody or antigen binding fragment having such an amino acidsequence encompasses, for example, a humanized antibody or antigenbinding fragment.

As used herein the term “from one to twenty-five” includes everyindividual values and ranges such as for example, 1, 2, 3, and up to 25;1 to 25; 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19; 1 to 18;1 to 17; 1 to 16; 1 to 15 and so on; 2 to 25, 2 to 24, 2 to 23, 2 to 22,2 to 21, 2 to 20; 2 to 19; 2 to 18; 2 to 17 and so on; 3 to 25, 3 to 24,3 to 23, 3 to 22, 3 to 21, 3 to 20; 3 to 19; 3 to 18 and so on; 4 to 25,4 to 24, 4 to 23, 4 to 22, 4 to 21, 4 to 20; 4 to 19; 4 to 18; 4 to 17;4 to 16 and so on; 5 to 25, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20;5 to 19; 5 to 18; 5 to 17 and so on, etc.

As used herein the term “from one to twenty-three” includes everyindividual values and ranges such as for example, 1, 2, 3, and up to 23;1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19; 1 to 18; 1 to 17; 1 to 16;1 to 15 and so on; 2 to 23, 2 to 22, 2 to 21, 2 to 20; 2 to 19; 2 to 18;2 to 17 and so on; 3 to 23, 3 to 22, 3 to 21, 3 to 20; 3 to 19; 3 to 18and so on; 4 to 23, 4 to 22, 4 to 21, 4 to 20; 4 to 19; 4 to 18; 4 to17; 4 to 16 and so on; 5 to 25, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to20; 5 to 19; 5 to 18; 5 to 17 and so on, etc.

As used herein the term “from one to twenty” includes every individualvalues and ranges such as for example, 1, 2, 3, and up to 20; 1 to 20; 1to 19; 1 to 18; 1 to 17; 1 to 16; 1 to 15 and so on; 2 to 20; 2 to 19; 2to 18; 2 to 17 and so on; 3 to 20; 3 to 19; 3 to 18 and so on; 4 to 20;4 to 19; 4 to 18; 4 to 17; 4 to 16 and so on; 5 to 20; 5 to 19; 5 to 18;5 to 17 and so on, etc.

Likewise, the term “from one to fifteen” includes every individualvalues and ranges such as for example, 1, 2, 3, and up to 15; 1 to 15; 1to 14; 1 to 13; 1 to 12; 1 to 11; 1 to 10 and so on; 2 to 15; 2 to 14; 2to 13; 2 to 12 and so on; 3 to 15; 3 to 14; 3 to 13 and so on; 4 to 15;4 to 14; 4 to 13; 4 to 12; 4 to 11 and so on; 5 to 15; 5 to 14; 5 to 13;5 to 12 and so on, etc.

Likewise, the term “from one to eleven” includes every individual valuesand ranges such as for example, 1, 2, 3, and up to 11; 1 to 11; 1 to 10,1 to 9, 1 to 8, 1 to 7, and so on; 2 to 11; 2 to 10; 2 to 9; 2 to 8 andso on; 3 to 11; 3 to 10; 3 to 9 and so on; 4 to 11; 4 to 10; 4 to 9; 4to 8; 4 to 7 and so on; 5 to 11; 5 to 10; 5 to 9; 5 to 8 and so on, etc.

In a more specific embodiment of the invention, the number of amino acidsubstitutions that may be accommodated in a humanized light chainvariable region derived from SEQ ID NO.:16 may be for example, from 1 to15 amino acid substitutions.

In yet a more specific embodiment of the invention, the number of aminoacid substitutions that may be accommodated in a humanized heavy chainvariable region derived from SEQ ID NO.:18 may be for example, from 1 to20 amino acid substitutions. In some instances, when considering ahumanized version of SEQ ID NO.:18, it may be useful to have at leastthree amino acid substitutions.

In a further more specific embodiment of the invention, the number ofamino acid substitutions that may be accommodated in a humanized lightchain variable region derived from SEQ ID NO.:24 may be for example,from 1 to 16 amino acid substitutions.

In yet a further more specific embodiment of the invention, the numberof amino acid substitutions that may be accommodated in a humanizedheavy chain variable region of SEQ ID NO.:26 may be for example, from 1to 17 amino acid substitutions.

In a further more specific embodiment of the invention, the number ofamino acid substitutions that may be accommodated in a humanized lightchain variable region derived from SEQ ID NO.:48 may be for example,from 1 to 11 amino acid substitutions.

In yet a further more specific embodiment of the invention, the numberof amino acid substitutions that may be accommodated in a humanizedheavy chain variable region of SEQ ID NO.:46 may be for example, from 1to 23 amino acid substitutions.

In accordance with an embodiment of the invention, the one to twentyamino acid substitutions may be for example, in the light chain variableregion.

In accordance with an embodiment of the invention, the one to twentyamino acid substitutions may be for example, in the heavy chain variableregion.

A humanized antibody or antigen binding fragment may therefore have alight chain variable region having up to twenty amino acid substitutionsin comparison with SEQ ID NO.:16 or SEQ ID NO.:24 and may have a heavychain variable region having up to twenty amino acid substitutions incomparison with SEQ ID NO.:18 or SEQ ID NO.:26. A humanized antibody orantigen binding fragment may therefore have a light chain variableregion having up to twenty-five amino acid substitutions in comparisonwith SEQ ID NO.:48 and may have a heavy chain variable region having upto twenty-five amino acid substitutions in comparison with SEQ IDNO.:46.

It is to be understood herein that when the humanized antibody orantigen binding fragment has two light chain variable regions and twoheavy chain variable regions, each one of the light chain variableregions may independently have up to twenty-five, twenty-four,twenty-three, twenty-two, twenty-one, twenty, nineteen, eighteen,seventeen, sixteen, fifteen, fourteen, thirteen, twelve, eleven, ten,nine, eight, seven, six, five, four, three, two, one amino acidsubstitutions and each one of the heavy chain variable regions may haveup to twenty-five, twenty-four, twenty-three, twenty-two, twenty-one,twenty, nineteen, eighteen, seventeen, sixteen, fifteen, fourteen,thirteen, twelve, eleven, ten, nine, eight, seven, six, five, four,three, two, one amino acid substitutions.

As discussed herein the amino acid substitutions may be conservative ornon-conservative. In an exemplary embodiment the amino acidsubstitutions may be conservative.

It is to be understood herein that the humanized antibody or antigenbinding fragment of the invention may also have a light chain variableregion and/or heavy chain variable region showing a deletion incomparison with SEQ ID NO.:16, SEQ ID NO.:18, SEQ ID NO.:189, SEQ IDNO.:190, SEQ ID NO.:194, SEQ ID NO.:195, SEQ ID NO.:196, SEQ ID NO.:197,SEQ ID NO.:24 and/or SEQ ID NO.:26. Such deletion may be found, forexample, at an amino- or carboxy-terminus of the light chain variableregion and/or heavy chain variable region.

Another exemplary embodiment of the humanized antibody or antigenbinding fragment of the present invention includes for example, anantibody or antigen binding fragment having a light chain variableregion which may comprise at least 90 consecutive amino acids of any ofSEQ ID NO.:186, SEQ ID NO.:187, SEQ ID NO.:188, SEQ ID NO.:189 or SEQ IDNO.:190.

As used herein the term “at least 90 consecutive amino acids of SEQ IDNO.:186” also includes the terms “at least 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, orat least 112 consecutive amino acids”. The term “at least 90 consecutiveamino acids of SEQ ID NO.:186” encompasses any possible sequence of atleast 90 consecutive amino acids found in SEQ ID NO.:186 and especiallythose sequences which include the 3 CDRs of SEQ ID NO.:186, such as, forexample a sequence comprising amino acids 6 to 108, 5 to 109, 13 to 103,14 to 111 of SEQ ID NO.:186 and so on.

As used herein the term “at least 90 consecutive amino acids of SEQ IDNO.:187” also includes the terms “at least 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, orat least 112 consecutive amino acids”. The term “at least 90 consecutiveamino acids of SEQ ID NO.:187” encompasses any possible sequence of atleast 90 consecutive amino acids found in SEQ ID NO.:187 and especiallythose sequences which include the 3 CDRs of SEQ ID NO.:187, such as, forexample a sequence comprising amino acids 7 to 109, 12 to 104, 22 to113, 18 to 112 of SEQ ID NO.:187 and so on.

The terms “at least 90 consecutive amino acids of SEQ ID NO.:188”, “atleast 90 consecutive amino acids of SEQ ID NO.:189” or “at least 90consecutive amino acids of SEQ ID NO.:190” has a similar meaning.

In accordance with the present invention, the antibody or antigenbinding fragment of the present invention may have, for example, a lightchain variable region as set forth in SEQ ID NO.:189 or 190.

The humanized antibody or antigen binding fragment of the inventionincludes (or further includes) for example, a heavy chain variableregion which may comprise at least 90 consecutive amino acids of any ofSEQ ID NOs.:191, 192, 193, 194, 195, 196 or 197.

As used herein the term “at least 90 consecutive amino acids of SEQ IDNO.:191” also includes the terms “at least 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115 or at least 116 consecutive amino acids”. The term “atleast 90 consecutive amino acids of SEQ ID NO.:191” encompasses anypossible sequence of at least 90 consecutive amino acids found in SEQ IDNO.:191 and especially those sequences which include the 3 CDRs of SEQID NO.:191, such as, for example a sequence comprising amino acids 1 to106, 2 to 112, 11 to 113, 7 to 102 of SEQ ID NO.:191 and so on.

As used herein the term “at least 90 consecutive amino acids of SEQ IDNO.:192” also includes the terms “at least 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115 or at least 116 consecutive amino acids”. The term “atleast 90 consecutive amino acids of SEQ ID NO.:192” encompasses anypossible sequence of at least 90 consecutive amino acids found in SEQ IDNO.:192 and especially those sequences which include the 3 CDRs of SEQID NO.:192, for example a sequence comprising amino acids 6 to 109, 8 to113, 1 to 102, 2 to 105 of SEQ ID NO.:192 and so on.

The terms “at least 90 consecutive amino acids of SEQ ID NO.:193”, “atleast 90 consecutive amino acids of SEQ ID NO.:194”, “at least 90consecutive amino acids of SEQ ID NO.:195”, “at least 90 consecutiveamino acids of SEQ ID NO.:196” or “at least 90 consecutive amino acidsof SEQ ID NO.:197” has a similar meaning.

In accordance with the present invention, the antibody or antigenbinding fragment of the present invention may have, for example, a heavychain variable region as set forth in SEQ ID NO.:194, 195, 196 or 197.

In accordance with the present invention the antibody or antigen bindingfragment may comprise, for example,

-   -   a) a light chain variable region which may comprise at least 90        consecutive amino acids of SEQ ID NO.:186 and a heavy chain        variable region which may comprise at least 90 consecutive amino        acids of any of SEQ ID NO.:191, SEQ ID NO.:192, SEQ ID NO.:193,        SEQ ID NO.:194, SEQ ID NO.:195, SEQ ID NO.:196 or SEQ ID        NO.:197;    -   b) a light chain variable region which may comprise at least 90        consecutive amino acids of SEQ ID NO.:187 and a heavy chain        variable region which may comprise at least 90 consecutive amino        acids of any of SEQ ID NO.:191, SEQ ID NO.:192, SEQ ID NO.:193,        SEQ ID NO.:194, SEQ ID NO.:195, SEQ ID NO.:196 or SEQ ID        NO.:197;    -   c) a light chain variable region which may comprise amino acids        at least 90 consecutive amino acids of SEQ ID NO.:188 and a        heavy chain variable region which may comprise at least 90        consecutive amino acids of any of SEQ ID NO.:191, SEQ ID        NO.:192, SEQ ID NO.:193, SEQ ID NO.:194, SEQ ID NO.:195, SEQ ID        NO.:196 or SEQ ID NO.:197;    -   d) a light chain variable region which may comprise at least 90        consecutive amino acids of SEQ ID NO.:189 and a heavy chain        variable region which may comprise at least 90 consecutive amino        acids of any of SEQ ID NO.:191, SEQ ID NO.:192, SEQ ID NO.:193,        SEQ ID NO.:194, SEQ ID NO.:195, SEQ ID NO.:196 or SEQ ID NO.:197        or    -   e) a light chain variable region which may comprise at least 90        consecutive amino acids of SEQ ID NO.:190 and a heavy chain        variable region which may comprise at least 90 consecutive amino        acids of any of SEQ ID NO.:191, SEQ ID NO.:192, SEQ ID NO.:193,        SEQ ID NO.:194, SEQ ID NO.:195, SEQ ID NO.:196 or SEQ ID        NO.:197.

In accordance with a more specific embodiment of the invention, thelight chain variable region may comprise at least 90 consecutive aminoacids of SEQ ID NO.:189 or 190 and the heavy chain variable region maycomprise at least 90 consecutive amino acids of SEQ ID NO.:194, 195, 196or 197.

In accordance with an even more specific embodiment of the invention,the light chain variable region may be as set forth in SEQ ID NO.:189and the heavy chain variable region may be as set forth in SEQ IDNO.:194.

In accordance with an even more specific embodiment of the invention,the light chain variable region may be as set forth in SEQ ID NO.:189and the heavy chain variable region may be as set forth in SEQ IDNO.:195.

In accordance with an even more specific embodiment of the invention,the light chain variable region may be as set forth in SEQ ID NO.:189and the heavy chain variable region may be as set forth in SEQ IDNO.:196.

In accordance with an even more specific embodiment of the invention,the light chain variable region may be as set forth in SEQ ID NO.:189and the heavy chain variable region may be as set forth in SEQ IDNO.:197.

In accordance with an even more specific embodiment of the invention,the light chain variable region may be as set forth in SEQ ID NO.:190and the heavy chain variable region may be as set forth in SEQ IDNO.:194.

In accordance with an even more specific embodiment of the invention,the light chain variable region may be as set forth in SEQ ID NO.:190and the heavy chain variable region may be as set forth in SEQ IDNO.:195.

In accordance with an even more specific embodiment of the invention,the light chain variable region may be as set forth in SEQ ID NO.:190and the heavy chain variable region may be as set forth in SEQ IDNO.:196.

In accordance with an even more specific embodiment of the invention,the light chain variable region may be as set forth in SEQ ID NO.:190and the heavy chain variable region may be as set forth in SEQ IDNO.:197.

Another exemplary embodiment of the humanized antibody or antigenbinding fragment of the present invention includes for example, anantibody or antigen binding fragment having a light chain variableregion which may comprise at least 90 consecutive amino acids of any ofSEQ ID NO.:174, SEQ ID NO.:175, SEQ ID NO.:176 or SEQ ID NO.:168.

As used herein the term “at least 90 consecutive amino acids of SEQ IDNO.:174” also includes the terms “at least 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112or at least 113 consecutive amino acids”. The term “at least 90consecutive amino acids of SEQ ID NO.:174” encompasses any possiblesequence of at least 90 consecutive amino acids found in SEQ ID NO.:174and especially those sequences which include the 3 CDRs of SEQ IDNO.:174, such as, for example a sequence comprising amino acids 6 to108, 5 to 109, 13 to 103, 14 to 111 of SEQ ID NO.:174 and so on.

As used herein the term “at least 90 consecutive amino acids of SEQ IDNO.:175” also includes the terms “at least 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112or at least 113 consecutive amino acids”. The term “at least 90consecutive amino acids of SEQ ID NO.:175” encompasses any possiblesequence of at least 90 consecutive amino acids found in SEQ ID NO.:175and especially those sequences which include the 3 CDRs of SEQ IDNO.:175, such as, for example a sequence comprising amino acids 7 to109, 12 to 104, 22 to 113, 18 to 112 of SEQ ID NO.:175 and so on.

The terms “at least 90 consecutive amino acids of SEQ ID NO.:176” or “atleast 90 consecutive amino acids of SEQ ID NO.:168” has a similarmeaning.

In accordance with the present invention, the antibody or antigenbinding fragment of the present invention may have, for example, a lightchain variable region as set forth in SEQ ID NO.:168.

The humanized antibody or antigen binding fragment of the inventionincludes (or further includes) for example, a heavy chain variableregion which may comprise at least 90 consecutive amino acids of any ofSEQ ID NOs.:177, 178, 179 or 169.

As used herein the term “at least 90 consecutive amino acids of SEQ IDNO.:177” also includes the terms “at least 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112or at least 113 consecutive amino acids”. The term “at least 90consecutive amino acids of SEQ ID NO.:177” encompasses any possiblesequence of at least 90 consecutive amino acids found in SEQ ID NO.:177and especially those sequences which include the 3 CDRs of SEQ IDNO.:177, such as, for example a sequence comprising amino acids 1 to106, 2 to 112, 11 to 113, 7 to 102 of SEQ ID NO.:177 and so on.

As used herein the term “at least 90 consecutive amino acids of SEQ IDNO.:178” also includes the terms “at least 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112or at least 113 consecutive amino acids”. The term “at least 90consecutive amino acids of SEQ ID NO.:178” encompasses any possiblesequence of at least 90 consecutive amino acids found in SEQ ID NO.:178and especially those sequences which include the 3 CDRs of SEQ IDNO.:178, for example a sequence comprising amino acids 6 to 109, 8 to113, 1 to 102, 2 to 105 of SEQ ID NO.:178 and so on.

The terms “at least 90 consecutive amino acids of SEQ ID NO.:179” or “atleast 90 consecutive amino acids of SEQ ID NO.:169” has a similarmeaning.

In accordance with the present invention, the antibody or antigenbinding fragment of the present invention may have, for example, a heavychain variable region as set forth in SEQ ID NO.:169.

In accordance with the present invention the antibody or antigen bindingfragment may comprise, for example,

-   -   f) a light chain variable region which may comprise at least 90        consecutive amino acids of SEQ ID NO.:174 and a heavy chain        variable region which may comprise at least 90 consecutive amino        acids of any of SEQ ID NO.:177, SEQ ID NO.:178, SEQ ID NO.:179        or SEQ ID NO.:169;    -   g) a light chain variable region which may comprise at least 90        consecutive amino acids of SEQ ID NO.:175 and a heavy chain        variable region which may comprise at least 90 consecutive amino        acids of any of SEQ ID NO.:177, SEQ ID NO.:178, SEQ ID NO.:179        or SEQ ID NO.:169;    -   h) a light chain variable region which may comprise amino acids        at least 90 consecutive amino acids of SEQ ID NO.:176 and a        heavy chain variable region which may comprise at least 90        consecutive amino acids of any of SEQ ID NO.:177, SEQ ID        NO.:178, SEQ ID NO.:179 or SEQ ID NO.:169 or;    -   i) a light chain variable region which may comprise at least 90        consecutive amino acids of SEQ ID NO.:168 and a heavy chain        variable region which may comprise at least 90 consecutive amino        acids of any of SEQ ID NO.:177, SEQ ID NO.:178, SEQ ID NO.:179        or SEQ ID NO.:169.

In accordance with a more specific embodiment of the invention, thelight chain variable region may comprise at least 90 consecutive aminoacids of SEQ ID NO.:168 and the heavy chain variable region may compriseat least 90 consecutive amino acids of SEQ ID NO.:169.

In accordance with an even more specific embodiment of the invention,the light chain variable region may be as set forth in SEQ ID NO.:168and the heavy chain variable region may be as set forth in SEQ IDNO.:169.

Other exemplary embodiments of the humanized antibodies or antigenbinding fragments of the invention are those which may comprise a lightchain variable region which may comprise at least 90 consecutive aminoacids of any of SEQ ID Nos. 180, 181, 182 or 172.

As used herein the term “at least 90 consecutive amino acids of SEQ IDNO.:180” also includes the terms “at least 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106 or at least 107, consecutiveamino acids”. The term “at least 90 consecutive amino acids of SEQ IDNO.:180” encompasses any possible sequence of at least 90 consecutiveamino acids found in SEQ ID NO.:180 and especially those sequences whichinclude the 3 CDRs of SEQ ID NO.:180, for example a sequence comprisingamino acids 6 to 102, 11 to 106, 1 to 106, 3 to 95, 5 to 95 of SEQ IDNO.:180 and so on.

As used herein the term “at least 90 consecutive amino acids of SEQ IDNO.:181” also includes the terms “at least 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106 or at least 107, consecutiveamino acids”. The term “at least 90 consecutive amino acids of SEQ IDNO.:181” encompasses any possible sequence of at least 90 consecutiveamino acids found in SEQ ID NO.:181 and especially those sequences whichinclude the 3 CDRs of SEQ ID NO.:181, for example a sequence comprisingamino acids 9 to 106, 10 to 101, 1 to 98, 3 to 99, 7 to 107 of SEQ IDNO.:181 and so on.

The terms “at least 90 consecutive amino acids of SEQ ID NO.:182” or “atleast 90 consecutive amino acids of SEQ ID NO.:172” has a similarmeaning.

In accordance with the present invention, the antibody or antigenbinding fragment of the present invention may have, for example, a lightchain variable region as set forth in SEQ ID NO.:172.

The humanized antibody or antigen binding fragment of the inventionincludes (or further includes) for example, a heavy chain variableregion which may comprise at least 90 consecutive amino acids of any ofSEQ ID NOs.:183, 184, 185 or 173.

As used herein the term “at least 90 consecutive amino acids of SEQ IDNO.:183” also includes the terms “at least 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115 or at least 116 consecutive amino acids”. The term “atleast 90 consecutive amino acids of SEQ ID NO.:183” encompasses anypossible sequence of at least 90 consecutive amino acids found in SEQ IDNO.:183 and especially those sequences which include the 3 CDRs of SEQID NO.:183, such as, for example a sequence comprising amino acids 6 to111, 1 to 106, 2 to 104, 5 to 106, 10 to 107 of SEQ ID NO.:183 and soon.

As used herein the term “at least 90 consecutive amino acids of SEQ IDNO.:185” also includes the terms “at least 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115 or at least 116 consecutive amino acids”. The term “atleast 90 consecutive amino acids of SEQ ID NO.:185” encompasses anypossible sequence of at least 90 consecutive amino acids found in SEQ IDNO.:185 and especially those sequences which include the 3 CDRs of SEQID NO.:185, such as, for example a sequence comprising amino acids 3 to107, 1 to 115, 1 to 110, 22 to 116, 20 to 115 of SEQ ID NO.:185 and soon.

The terms “at least 90 consecutive amino acids of SEQ ID NO.:184” or “atleast 90 consecutive amino acids of SEQ ID NO.:173” has a similarmeaning.

In accordance with the present invention, the antibody or antigenbinding fragment of the present invention may have, for example, a heavychain variable region as set forth in SEQ ID NO.:173.

In accordance with the present invention the antibody or antigen bindingfragment may comprise, for example,

-   -   a) a light chain variable region which may comprise at least 90        consecutive amino acids of SEQ ID NO.:180 and a heavy chain        variable region which may comprise at least 90 consecutive amino        acids of any of SEQ ID NO.:183, SEQ ID NO.:184, SEQ ID NO.:185        or SEQ ID NO.:173;    -   b) a light chain variable region which may comprise at least 90        consecutive amino acids of SEQ ID NO.:181 and a heavy chain        variable region which may comprise at least 90 consecutive amino        acids of any of SEQ ID NO.:183, SEQ ID NO.:184, SEQ ID NO.:185        or SEQ ID NO.:173;    -   c) a light chain variable region which may comprise amino acids        at least 90 consecutive amino acids of SEQ ID NO.:182 and a        heavy chain variable region which may comprise at least 90        consecutive amino acids of any of SEQ ID NO.:183, SEQ ID        NO.:184, SEQ ID NO.:185 or SEQ ID NO.:173 or;    -   d) a light chain variable region which may comprise at least 90        consecutive amino acids of SEQ ID NO.:172 and a heavy chain        variable region which may comprise at least 90 consecutive amino        acids of any of SEQ ID NO.:183, SEQ ID NO.:184, SEQ ID NO.:185        or SEQ ID NO.:173.

In accordance with a more specific embodiment of the invention, thelight chain variable region may have at least 90 consecutive amino acidsof SEQ ID NO.:172 and the heavy chain variable region may have at least90 consecutive amino acids of SEQ ID NO.:173.

In accordance with an even more specific embodiment of the invention,the light chain variable region may be as set forth in SEQ ID NO.:172and the heavy chain variable region may be as set forth in SEQ IDNO.:173.

The antibody or antigen binding fragment of the present invention mayhave a light chain variable region and/or heavy chain variable region asdescribed above and may further comprise amino acids of a constantregion, such as, for example, amino acids of a constant region of ahuman antibody.

In an exemplary embodiment, the antibody or antigen binding fragment ofthe present invention may comprise, for example, a human IgG1 constantregion.

In accordance with another exemplary embodiment of the invention, theantigen binding fragment may be, for example, a scFv, a Fab, a Fab′ or a(Fab)₂.

Production of the Antibodies in Cells

The anti-KAAG1 antibodies that are disclosed herein can be made by avariety of methods familiar to those skilled in the art, such ashybridoma methodology or by recombinant DNA methods.

In an exemplary embodiment of the invention, the anti-KAAG1 antibodiesmay be produced by the conventional hybridoma technology, where a mouseis immunized with an antigen, spleen cells isolated and fused withmyeloma cells lacking HGPRT expression and hybrid cells selected byhypoxanthine, aminopterin and thymine (HAT) containing media.

In an additional exemplary embodiment of the invention, the anti-KAAG1antibodies may be produced by recombinant DNA methods.

In order to express the anti-KAAG1 antibodies, nucleotide sequences ableto encode any one of a light and heavy immunoglobulin chains describedherein or any other may be inserted into an expression vector, i.e., avector that contains the elements for transcriptional and translationalcontrol of the inserted coding sequence in a particular host. Theseelements may include regulatory sequences, such as enhancers,constitutive and inducible promoters, and 5′ and 3′ un-translatedregions. Methods that are well known to those skilled in the art may beused to construct such expression vectors. These methods include invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination.

A variety of expression vector/host cell systems known to those of skillin the art may be utilized to express a polypeptide or RNA derived fromnucleotide sequences able to encode any one of a light and heavyimmunoglobulin chains described herein. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith baculovirus vectors; plant cell systems transformed with viral orbacterial expression vectors; or animal cell systems. For long-termproduction of recombinant proteins in mammalian systems, stableexpression in cell lines may be effected. For example, nucleotidesequences able to encode any one of a light and heavy immunoglobulinchains described herein may be transformed into cell lines usingexpression vectors that may contain viral origins of replication and/orendogenous expression elements and a selectable or visible marker geneon the same or on a separate vector. The invention is not to be limitedby the vector or host cell employed. In certain embodiments of thepresent invention, the nucleotide sequences able to encode any one of alight and heavy immunoglobulin chains described herein may each beligated into a separate expression vector and each chain expressedseparately. In another embodiment, both the light and heavy chains ableto encode any one of a light and heavy immunoglobulin chains describedherein may be ligated into a single expression vector and expressedsimultaneously.

Alternatively, RNA and/or polypeptide may be expressed from a vectorcomprising nucleotide sequences able to encode any one of a light andheavy immunoglobulin chains described herein using an in vitrotranscription system or a coupled in vitro transcription/translationsystem respectively.

In general, host cells that contain nucleotide sequences able to encodeany one of a light and heavy immunoglobulin chains described hereinand/or that express a polypeptide encoded by the nucleotide sequencesable to encode any one of a light and heavy immunoglobulin chainsdescribed herein, or a portion thereof, may be identified by a varietyof procedures known to those of skill in the art. These proceduresinclude, but are not limited to, DNA/DNA or DNA/RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques thatinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or amino acid sequences.Immunological methods for detecting and measuring the expression ofpolypeptides using either specific polyclonal or monoclonal antibodiesare known in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). Those of skill in the artmay readily adapt these methodologies to the present invention.

Host cells comprising nucleotide sequences able to encode any one of alight and heavy immunoglobulin chains described herein may thus becultured under conditions for the transcription of the corresponding RNA(mRNA, siRNA, shRNA etc.) and/or the expression of the polypeptide fromcell culture. The polypeptide produced by a cell may be secreted or maybe retained intracellularly depending on the sequence and/or the vectorused. In an exemplary embodiment, expression vectors containingnucleotide sequences able to encode any one of a light and heavyimmunoglobulin chains described herein may be designed to contain signalsequences that direct secretion of the polypeptide through a prokaryoticor eukaryotic cell membrane.

Due to the inherent degeneracy of the genetic code, other DNA sequencesthat encode the same, substantially the same or a functionallyequivalent amino acid sequence may be produced and used, for example, toexpress a polypeptide encoded by nucleotide sequences able to encode anyone of a light and heavy immunoglobulin chains described herein. Thenucleotide sequences of the present invention may be engineered usingmethods generally known in the art in order to alter the nucleotidesequences for a variety of purposes including, but not limited to,modification of the cloning, processing, and/or expression of the geneproduct. DNA shuffling by random fragmentation and PCR reassembly ofgene fragments and synthetic oligonucleotides may be used to engineerthe nucleotide sequences. For example, oligonucleotide-mediatedsite-directed mutagenesis may be used to introduce mutations that createnew restriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, and so forth.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed polypeptide in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation. In anexemplary embodiment, anti-KAAG1 antibodies that contain particularglycosylation structures or patterns may be desired. Post-translationalprocessing, which cleaves a “prepro” form of the polypeptide, may alsobe used to specify protein targeting, folding, and/or activity.Different host cells that have specific cellular machinery andcharacteristic mechanisms for post-translational activities (e.g., CHO,HeLa, MDCK, HEK293, and W138) are available commercially and from theAmerican Type Culture Collection (ATCC) and may be chosen to ensure thecorrect modification and processing of the expressed polypeptide.

Those of skill in the art will readily appreciate that natural,modified, or recombinant nucleic acid sequences may be ligated to aheterologous sequence resulting in translation of a fusion polypeptidecontaining heterologous polypeptide moieties in any of theaforementioned host systems. Such heterologous polypeptide moieties mayfacilitate purification of fusion polypeptides using commerciallyavailable affinity matrices. Such moieties include, but are not limitedto, glutathione S-transferase (GST), maltose binding protein,thioredoxin, calmodulin binding peptide, 6-His (His), FLAG, c-myc,hemaglutinin (HA), and antibody epitopes such as monoclonal antibodyepitopes.

In yet a further aspect, the present invention relates to apolynucleotide which may comprise a nucleotide sequence encoding afusion protein. The fusion protein may comprise a fusion partner (e.g.,HA, Fc, etc.) fused to the polypeptide (e.g., complete light chain,complete heavy chain, variable regions, CDRs etc.) described herein.

Those of skill in the art will also readily recognize that the nucleicacid and polypeptide sequences may be synthesized, in whole or in part,using chemical or enzymatic methods well known in the art. For example,peptide synthesis may be performed using various solid-phase techniquesand machines such as the ABI 431A Peptide synthesizer (PE Biosystems)may be used to automate synthesis. If desired, the amino acid sequencemay be altered during synthesis and/or combined with sequences fromother proteins to produce a variant protein.

Antibody Conjugates

The antibody or antigen binding fragment of the present invention may beconjugated with a detectable moiety (i.e., for detection or diagnosticpurposes) or with a therapeutic moiety (for therapeutic purposes)

A “detectable moiety” is a moiety detectable by spectroscopic,photochemical, biochemical, immunochemical, chemical and/or otherphysical means. A detectable moiety may be coupled either directlyand/or indirectly (for example via a linkage, such as, withoutlimitation, a DOTA or NHS linkage) to antibodies and antigen bindingfragments thereof of the present invention using methods well known inthe art. A wide variety of detectable moieties may be used, with thechoice depending on the sensitivity required, ease of conjugation,stability requirements and available instrumentation. A suitabledetectable moiety include, but is not limited to, a fluorescent label, aradioactive label (for example, without limitation, ¹²⁵I, In¹¹¹, Tc⁹⁹,I¹³¹ and including positron emitting isotopes for PET scanner etc), anuclear magnetic resonance active label, a luminiscent label, achemiluminescent label, a chromophore label, an enzyme label (forexample and without limitation horseradish peroxidase, alkalinephosphatase, etc.), quantum dots and/or a nanoparticle. Detectablemoiety may cause and/or produce a detectable signal thereby allowing fora signal from the detectable moiety to be detected.

In another exemplary embodiment of the invention, the antibody orantigen binding fragment thereof may be coupled (modified) with atherapeutic moiety (e.g., drug, cytotoxic moiety).

In an exemplary embodiment, the anti-KAAG1 antibodies and antigenbinding fragments may comprise an inhibitor, a chemotherapeutic orcytotoxic agent. For example, the antibody and antigen binding fragmentsmay be conjugated to the chemotherapeutic or cytotoxic agent. Suchchemotherapeutic or cytotoxic agents include, but are not limited to,Yttrium-90, Scandium-47, Rhenium-186, Iodine-131, Iodine-125, and manyothers recognized by those skilled in the art (e.g., lutetium (e.g.,Lu¹⁷⁷), bismuth (e.g., Bi²¹³), copper (e.g., Cu⁶⁷)). In other instances,the chemotherapeutic or cytotoxic agent may comprise, withoutlimitation, 5-fluorouracil, adriamycin, irinotecan, platinum-basedcompounds such as cisplatin and anti-tubulin or anti-mitotic compoundssuch as, taxanes, doxorubicin and cyclophosphamide, pseudomonasendotoxin, ricin and other toxins. Suitable antibody drug conjugates areselected amongst those having an IC₅₀ in the range of 0.001 nM to 150nM, 0.001 nM to 100 nM, 0.001 nM to 50 nM, 0.001 nM to 20 nM or 0.001 nMto 10 nM (inclusively). The cytotoxic drug used for conjugation is thusselected on the basis of these criteria.

Alternatively, in order to carry out the methods of the presentinvention and as known in the art, the antibody or antigen bindingfragment of the present invention (conjugated or not) may be used incombination with a second molecule (e.g., a secondary antibody, etc.)which is able to specifically bind to the antibody or antigen bindingfragment of the present invention and which may carry a desirabledetectable, diagnostic or therapeutic moiety.

Pharmaceutical Compositions of the Antibodies and their Use

Pharmaceutical compositions of the anti-KAAG1 antibodies or antigenbinding fragments (conjugated or not) are also encompassed by thepresent invention. The pharmaceutical composition may comprise ananti-KAAG1 antibody or an antigen binding fragment and may also containa pharmaceutically acceptable carrier.

Other aspects of the invention relate to a composition which maycomprise the antibody or antigen binding fragment described herein and acarrier.

The present invention also relates to a pharmaceutical composition whichmay comprise the antibody or antigen binding fragment described hereinand a pharmaceutically acceptable carrier.

In addition to the active ingredients, a pharmaceutical composition maycontain pharmaceutically acceptable carriers comprising water, PBS, saltsolutions, gelatins, oils, alcohols, and other excipients andauxiliaries that facilitate processing of the active compounds intopreparations that may be used pharmaceutically. In other instances, suchpreparations may be sterilized.

As used herein, “pharmaceutical composition” means therapeuticallyeffective amounts of the agent together with pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvant and/orcarriers. A “therapeutically effective amount” as used herein refers tothat amount which provides a therapeutic effect for a given conditionand administration regimen. Such compositions are liquids or lyophilizedor otherwise dried formulations and include diluents of various buffercontent (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts).Solubilizing agents (e.g., glycerol, polyethylene glycerol),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., thimerosal, benzyl alcohol, parabens), bulking substances ortonicity modifiers (e.g., lactose, mannitol), covalent attachment ofpolymers such as polyethylene glycol to the protein, complexation withmetal ions, or incorporation of the material into or onto particulatepreparations of polymeric compounds such as polylactic acid,polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils). Alsocomprehended by the invention are particulate compositions coated withpolymers (e.g., poloxamers or poloxamines). Other embodiments of thecompositions of the invention incorporate particulate forms protectivecoatings, protease inhibitors or permeation enhancers for various routesof administration, including parenteral, pulmonary, nasal, oral,vaginal, rectal routes. In one embodiment the pharmaceutical compositionis administered parenterally, paracancerally, transmucosally,transdermally, intramuscularly, intravenously, intradermally,subcutaneously, intraperitonealy, intraventricularly, intracranially andintratumorally.

Further, as used herein “pharmaceutically acceptable carrier” or“pharmaceutical carrier” are known in the art and include, but are notlimited to, 0.01-0.1 M or 0.05 M phosphate buffer or 0.8% saline.Additionally, such pharmaceutically acceptable carriers may be aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's orfixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, collating agents, inertgases and the like.

For any compound, the therapeutically effective dose may be estimatedinitially either in cell culture assays or in animal models such asmice, rats, rabbits, dogs, or pigs. An animal model may also be used todetermine the concentration range and route of administration. Suchinformation may then be used to determine useful doses and routes foradministration in humans. These techniques are well known to one skilledin the art and a therapeutically effective dose refers to that amount ofactive ingredient that ameliorates the symptoms or condition.Therapeutic efficacy and toxicity may be determined by standardpharmaceutical procedures in cell cultures or with experimental animals,such as by calculating and contrasting the ED₅₀ (the dosetherapeutically effective in 50% of the population) and LD₅₀ (the doselethal to 50% of the population) statistics. Any of the therapeuticcompositions described above may be applied to any subject in need ofsuch therapy, including, but not limited to, mammals such as dogs, cats,cows, horses, rabbits, monkeys, and humans.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

Methods of Use

The term “treatment” for purposes of this disclosure refers to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) the targeted pathologiccondition or disorder. Those in need of treatment include those alreadyhaving the disorder as well as those prone to have the disorder or thosein whom the disorder is to be prevented.

The present invention provides in one aspect thereof, a method oftreating an individual having or suspected of having breast cancer withan antibody or antigen binding fragment which is capable of specificbinding to KAAG1.

In accordance with the present invention, the individual may have abreast cancer that is negative for the estrogen receptor expression, theprogesterone receptor expression and/or Her2 expression (oroverexpression).

Also in accordance with the present invention, the individual may have abreast cancer that has low expression for at least one of estrogenreceptor, progesterone receptor and/or Her2.

For example, the tumor may be negative for (or have low expression of)both estrogen receptor expression and progesterone receptor expression.

In accordance with the present invention, the individual may have abreast cancer that is characterized as being triple-negative orbasal-like.

Yet other aspects of the invention relate to the use of the isolatedantibody or antigen binding fragment described herein in the treatmentor diagnosis of breast cancer characterized by a lack of estrogenreceptor expression, progesterone receptor expression and/or Her2overexpression or by low expression of at least one of these threemarkers.

In accordance with the present invention, the method may comprise, forexample, administering an antibody or antigen binding fragment which iscapable of specific binding to KAAG1 to an individual in need. Theindividual in need is preferentially selected on the basis of a lack ofER expression, PgR expression and/or by the absence of HER2 proteinover-expression. Clinical testing for these markers is usually performedusing histopathologic methods (immunohistochemistry, FISH, etc.) and/orby gene expression studies (see for example Dent et al, 2007, Bernsteinand Lacey, 2011). The individual in need may thus be an individual whohas received a diagnosis of triple-negative breast cancer or basal-likebreast cancer.

The present invention thus particularly relates to the therapeutictreatment of individual having triple-negative breast cancer orbasal-like cancer with an anti-KAAG1 antibody.

Suitable antibodies or antigen binding fragments include those that arecapable of specific binding to KAAG1 at the surface of tumor cells. Suchantibodies may preferentially bind an epitope included within aminoacids 30 to 84 of KAAG1 inclusively (e.g., within amino acids 36 to 60(inclusively) or within amino acids 61 to 84 (inclusively) of KAAG1).

Suitable antibodies may be those which mediate antibody-dependent cellcytotoxicity and those that are conjugated with a therapeutic moiety.

In accordance with the present invention, the antibody may be, forexample, a monoclonal antibody, a chimeric antibody or a humanizedantibody or an antigen binding fragment thereof.

The method of the present invention may include administering theantibody or antigen binding fragment in combination with an inhibitor, achemotherapeutic or a cytotoxic agent.

Other methods of treatment encompassed by the present invention includeadministering other types of KAAG1 inhibitors such as antisense-basedtherapeutics (siRNA, antisenses, ribozymes, etc.).

The present invention thus provides a method of treating triple-negativebreast cancer or basal-like breast cancer by administering an inhibitorof KAAG1 activity or expression to an individual in need.

The inhibitor may comprise a nucleotide sequence complementary to SEQ IDNO.:1 or to a fragment thereof. More particularly, the inhibitor maycomprise a nucleotide sequence complementary to nucleotides 738 to 992(inclusively) of SEQ ID NO.:1 or to a fragment thereof. For example, theinhibitor may include at least 10 consecutive nucleotides (at least 15,at least 20) which are complementary to SEQ ID NO.:1 or to nucleotides738 to 992 (inclusively) of SEQ ID NO.:1.

In certain instances, the anti-KAAG1 antibodies and fragments mayinteract with cancer cells that express KAAG1 and induce animmunological reaction by mediating ADCC. In other instances, theanti-KAAG1 antibodies and fragments may block the interaction of KAAG1with its protein partners.

In certain instances, the anti-KAAG1 antibodies and antigen bindingfragments thereof may be administered concurrently with other treatmentsgiven for the same condition (inhibitors, chemotherapeutics or cytotoxicagents). As such, the antibodies may be administered with a PARP1inhibitor, a EGFR inhibitor, anti-mitotics (eg., taxanes),platinum-based agents (eg., cisplatin), DNA damaging agents (eg.Doxorubicin) and other anti-cancer therapies that are known to thoseskilled in the art. In other instances, the anti-KAAG1 antibodies andantigen binding fragments thereof may be administered with othertherapeutic antibodies. These include, but are not limited to,antibodies that target EGFR, CD-20, and Her2.

The present invention relates in a further aspect thereof to a methodfor inhibiting the growth of KAAG1-expressing cell that are estrogenreceptor-negative (ER−), progesterone receptor negative (PgR−) and/orthat lacks Her2 overexpression (Her2−), the method may comprisecontacting the cell with an effective amount of the antibody or antigenbinding fragment described herein.

The present invention also encompasses method of treating cancer orinhibiting the growth of a KAAG1 expressing cells that are estrogenreceptor-negative (ER−), progesterone receptor negative (PgR−) and/orthat lacks Her2 overexpression (Her2−), in a mammal, the method maycomprise administering the antibody or antigen binding fragmentdescribed herein to a mammal in need.

In further aspects, the present invention provides method of treatment,diagnostic methods and method of detection using the antibody or antigenbinding fragment of the present invention and the use of theseantibodies or antigen binding fragment in the manufacture of apharmaceutical composition or drug for such purposes.

Method of treatment encompassed by the present invention includesadministering an antibody or antigen binding fragment described hereinto a mammal in need, and especially to a patient having or susceptibleof having a cancer characterized as being estrogen receptor-negative(ER−), progesterone receptor negative (PgR−) and/or that lacks Her2overexpression (Her2−),

The invention also provides in further aspects, methods for reducingtumor spread, tumor invasion, tumor formation or for inducing tumorlysis, which may comprise administering an isolated antibody or antigenbinding fragment to a mammal in need.

The invention therefore relates to the use of the isolated antibody orantigen binding fragment described herein in the (manufacture of apharmaceutical composition for) treatment of cancer, reduction of tumorspread, tumor invasion, tumor formation or for inducing tumor lysis ofKAAG1-expressing tumor cells that are estrogen receptor-negative (ER−),progesterone receptor negative (PgR−) and/or that lacks Her2overexpression (Her2−).

The antibody or antigen binding fragment may more particularly beapplicable for malignant tumor including, for example, a malignant tumorhaving the ability to metastasize and/or tumor cells characterized byanchorage-independent growth. The antibody or antigen binding fragmentof the present invention may also be used in the diagnosis of cancer.The diagnosis of cancer may be performed in vivo by administering theantibody or antigen binding fragment of the present invention to amammal having or suspected of having a cancer. The diagnosis may also beperformed ex vivo by contacting a sample obtained from the mammal withthe antibody or antigen binding fragment and determining the presence orabsence of cells (tumor cells) expressing KAAG1 or a KAAG1 variant.

The present invention also encompasses method of detecting cancer ordetecting a KAAG1 expressing cells that are estrogen receptor-negative(ER−), progesterone receptor negative (PgR−) and/or that lacks Her2overexpression (Her2−), in a mammal, the method may compriseadministering the antibody or antigen binding fragment described hereinto a mammal in need.

The present invention relates in another aspect thereof to a method fordetecting a cell expressing KAAG1 or a KAAG1 variant, the method maycomprise contacting the cell with an antibody or antigen bindingfragment described herein and detecting a complex formed by the antibodyand the KAAG1- or KAAG1 variant-expressing cell. Exemplary embodimentsof antibodies or antigen binding fragments used in detection methods arethose which are capable of binding to the extracellular region of KAAG1.

Other exemplary embodiments of antibodies or antigen binding fragmentsused in detection methods are those which bind to KAAG1 or KAAG1 variantexpressed at the surface of tumor cells that are estrogenreceptor-negative (ER−), progesterone receptor negative (PgR−) and/orthat lacks Her2 overexpression (Her2−).

Another aspect of the invention relates a method for detecting KAAG1(SEQ ID NO.:2), a KAAG1 variant having at least 80% sequence identitywith SEQ ID NO.:2 or a secreted form of circulating form of KAAG1 orKAAG1 variant, the method may comprise contacting a cell expressingKAAG1 or the KAAG1 variant or a sample (biopsy, serum, plasma, urineetc.) comprising or suspected of comprising KAAG1 or the KAAG1 variantwith the antibody or antigen binding fragments described herein andmeasuring binding. The sample may originate from a mammal (e.g., ahuman) which may have cancer (e.g., breast cancer that is characterizedas being estrogen receptor-negative (ER−), progesterone receptornegative (PgR−) and/or that lacks Her2 overexpression (Her2−), such asbasal-like breast cancer or triple-negative breast cancer) or may besuspected of having cancer. The sample may be a tissue sample obtainedfrom the mammal or a cell culture supernatant.

In accordance with the invention the sample may be a serum sample, aplasma sample, a blood sample or ascitic fluid obtained from the mammal.The antibody or antigen binding fragment described herein mayadvantageously detect a secreted or circulating form (circulating inblood) of KAAG1.

The method may comprise quantifying the complex formed by the antibodyor antigen binding fragment bound to KAAG1 or to the KAAG1 variant.

The binding of an antibody to an antigen will cause an increase in theexpected molecular weight of the antigen. A physical change thereforeoccurs upon specific binding of the antibody or antigen binding fragmentand the antigen.

Such changes may be detected using, for example, electrophoresisfollowed by Western blot and coloration of the gel or blot, massspectrometry, HPLC coupled with a computer or else. Apparatus capable ofcomputing a shift in molecular weight are known in the art and includefor example, Phosphorimager™.

When the antibody comprises for example a detectable label, theantigen-antibody complex may be detected by the fluorescence emitted bythe label, radiation emission of the label, enzymatic activity of alabel provided with its substrate or else.

Detection and/or measurement of binding between an antibody or antigenbinding fragment and an antigen may be performed by various methodsknown in the art. Binding between an antibody or antigen bindingfragment and an antigen may be monitored with an apparatus capable ofdetecting the signal emitted by the detectable label (radiationemission, fluorescence, color change etc.). Such apparatus provides datawhich indicates that binding as occurred and may also provide indicationas to the amount of antibody bound to the antigen. The apparatus(usually coupled with a computer) may also be capable of calculating thedifference between a background signal (e.g., signal obtained in theabsence of antigen-antibody binding) or background noise and the signalobtained upon specific antibody-antigen binding. Such apparatuses maythus provide the user with indications and conclusions as to whether theantigen has been detected or not.

Additional aspects of the invention relate to kits which may include oneor more container containing one or more antibodies or antigen bindingfragments described herein.

Nucleic Acids, Vectors and Cells

Antibodies are usually made in cells allowing expression of the lightchain and heavy chain expressed from a vector(s) comprising a nucleicacid sequence encoding the light chain and/or heavy chain.

The present therefore encompasses nucleic acids capable of encoding anyof the CDRs, light chain variable regions, heavy chain variable regions,light chains, heavy chains described herein.

The present invention therefore relates in a further aspect to a nucleicacid encoding a light chain variable region and/or a heavy chainvariable region of an antibody which is capable of specific binding toKAAG1.

Exemplary embodiments of nucleic acids encompassed by the presentinvention includes a nucleic acid selected from the group consisting ofa nucleic acid having at least 70% sequence identity (i.e., at least75%, at least 80% sequence identity) with any one of SEQ ID NOs.:3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 45 and 47, fragments (e.g., of atleast 10, at least 15, at least 20 consecutive nucleotides) andcomplement thereof.

In accordance with an embodiment of the invention, the nucleic acid mayespecially encode a light chain variable region and/or heavy chainvariable region of an antibody which may be capable of inducing killing(elimination, destruction, lysis) of KAAG1- or KAAG1 variant-expressingtumor cells.

In accordance with another embodiment of the invention, the nucleic acidmay especially encode a light chain variable region and/or heavy chainvariable region of an antibody which may be capable of reducingspreading of KAAG1- or KAAG1 variant-expressing tumor cells.

In accordance with yet another embodiment of the invention, the nucleicacid may particularly encode a light chain variable region and/or heavychain variable region of an antibody which may be capable of decreasingor impairing formation of KAAG1- or KAAG1 variant-expressing tumors.

Exemplary embodiments of nucleic acids of the present invention includenucleic acids encoding a light chain variable region comprising:

-   -   a. a CDRL1 sequence selected from the group consisting of SEQ ID        NO.:72 and SEQ ID NO.:73;    -   b. a CDRL2 sequence selected from the group consisting of SEQ ID        NO.:74, SEQ ID NO.: 75 and SEQ ID NO.:76, or;    -   c. a CDRL3 sequence selected from the group consisting of SEQ ID        NO.:77, SEQ ID NO.:78 and SEQ ID NO.:79.

In accordance with the present invention, the nucleic acid may encode alight chain variable region which may comprise at least two CDRs of aCDRL1, a CDRL2 or a CDRL3.

Also in accordance with the present invention, the nucleic acid mayencode a light chain variable region which may comprise one CDRL1, oneCDRL2 and one CDRL3.

The present invention also relates to a nucleic acid encoding a heavychain variable region comprising:

-   -   a. a CDRH1 sequence comprising SEQ ID NO.:80;    -   b. a CDRH2 sequence selected from the group consisting of SEQ ID        NO.:81, SEQ ID NO.:82, SEQ ID NO.:83, SEQ ID NO.:84 and SEQ ID        NO.:85, or;    -   c. a CDRH3 sequence selected from the group consisting of SEQ ID        NO.:86, SEQ ID NO.:87 and SEQ ID NO.:88.

In accordance with the present invention, the nucleic acid may encode aheavy chain variable region which may comprise at least two CDRs of aCDRH1, a CDRH2 or a CDRH3.

In accordance with the present invention, the nucleic acid may encode aheavy chain variable region which may comprise one CDRH1, one CDRH2 andone CDRH3.

Also encompassed by the present invention are nucleic acids encodingantibody variants having at least one conservative amino acidsubstitution.

In accordance with the present invention, the nucleic acid may encode aCDR comprising at least one conservative amino acid substitution.

In accordance with the present invention, the nucleic acid may encode aCDR comprising at least one conservative amino acid substitution in atleast two of the CDRs.

In accordance with the present invention, the nucleic acid may encode aCDR comprising at least one conservative amino acid substitution in the3 CDRs.

In accordance with the present invention, the nucleic acid may encode aCDR comprising at least two conservative amino acid substitutions in atleast one of the CDRs.

In accordance with the present invention, the nucleic acid may encode aCDR comprising at least two conservative amino acid substitutions in atleast two of the CDRs.

In accordance with the present invention, the nucleic acid may encode aCDR comprising at least two conservative amino acid substitutions in the3 CDRs.

Other aspects of the invention relate to a nucleic acid encoding a lightchain variable region having at least 70%, 75%, 80% sequence identitywith a sequence selected from the group consisting of SEQ ID NO.:16, SEQID NO.:20, SEQ ID NO.:24, SEQ ID NO.:103, SEQ ID NO.:104, SEQ IDNO.:105, SEQ ID NO.:106, SEQ ID NO.:107, SEQ ID NO.:108, SEQ ID NO.:109,SEQ ID NO.:110, SEQ ID NO.:111, SEQ ID NO.:112, SEQ ID NO.:113, SEQ IDNO.:114, SEQ ID NO.:115, SEQ ID NO.:116, SEQ ID NO.:117, SEQ ID NO.:118,SEQ ID NO.:119, SEQ ID NO.:120, SEQ ID NO.:121, SEQ ID NO.:122, SEQ IDNO.:123, SEQ ID NO.:124 and SEQ ID NO.:125.

Yet other aspects of the invention relate to a nucleic acid encoding aheavy chain variable region having at least 70%. 75%, 80% sequenceidentity to a sequence selected from the group consisting of SEQ IDNO.:18, SEQ ID NO.:22, SEQ ID NO.:26, SEQ ID NO.:126, SEQ ID NO.:127,SEQ ID NO.:128, SEQ ID NO.:129, SEQ ID NO.:130, SEQ ID NO.:131, SEQ IDNO.:132, SEQ ID NO.:133, SEQ ID NO.:134, SEQ ID NO.:135, SEQ ID NO.:136,SEQ ID NO.:137, SEQ ID NO.:138, SEQ ID NO.:139, SEQ ID NO.:140, SEQ IDNO.:141, SEQ ID NO.:142, SEQ ID NO.:143, SEQ ID NO.:144, SEQ ID NO.:145,SEQ ID NO.:146 and SEQ ID NO.:147.

In yet another aspect, the present invention relates to a vectorcomprising the nucleic acids described herein.

In accordance with the present invention, the vector may be anexpression vector.

Vector that contains the elements for transcriptional and translationalcontrol of the inserted coding sequence in a particular host are knownin the art. These elements may include regulatory sequences, such asenhancers, constitutive and inducible promoters, and 5′ and 3′un-translated regions. Methods that are well known to those skilled inthe art may be used to construct such expression vectors. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination.

In another aspect the present invention relates to an isolated cell thatmay comprise the nucleic acid described herein.

The isolated cell may comprise a nucleic acid encoding a light chainvariable region and a nucleic acid encoding a heavy chain variableregion either on separate vectors or on the same vector. The isolatedcell may also comprise a nucleic acid encoding a light chain and anucleic acid encoding a heavy chain either on separate vectors or on thesame vector.

In accordance with the present invention, the cell may be capable ofexpressing, assembling and/or secreting an antibody or antigen bindingfragment thereof.

In another aspect, the present invention provides a cell which maycomprise and/or may express the antibody described herein.

In accordance with the invention, the cell may comprise a nucleic acidencoding a light chain variable region and a nucleic acid encoding aheavy chain variable region.

The cell may be capable of expressing, assembling and/or secreting anantibody or antigen binding fragment thereof.

The examples below are presented to further outline details of thepresent invention.

EXAMPLES Example 1

This example discloses the methods used to convert the Fabs into fullIgG1 chimeric monoclonal antibodies.

Aside from the possibility of conducting interaction studies between theFab monoclonals and the KAAG1 protein, the use of Fabs may be limitedwith respect to conducting meaningful in vitro and in vivo studies tovalidate the biological function of the antigen. Thus, it was necessaryto transfer the light and heavy chain variable regions contained in theFabs to full antibody scaffolds, to generate mouse-human chimeric IgG1s.The expression vectors for both the light and heavy immunoglobulinchains were constructed such that i) the original bacterial signalpeptide sequences upstream of the Fab expression vectors were replacedby mammalian signal peptides and ii) the light and heavy chain constantregions in the mouse antibodies were replaced with human constantregions. The methods to accomplish this transfer utilized standardmolecular biology techniques that are familiar to those skilled in theart.

Light chain expression vector—an existing mammalian expression plasmid,called pTTVH8G (Durocher et al., 2002), designed to be used in the 293Etransient transfection system was modified to accommodate the mouselight chain variable region. The resulting mouse-human chimeric lightchain contained a mouse variable region followed by the human kappaconstant domain. The cDNA sequence encoding the human kappa constantdomain was amplified by PCR with primers OGS1773 and OGS1774 (SEQ IDNOS:55 and 56, respectively). The nucleotide sequence and thecorresponding amino acid sequence for the human kappa constant regionare shown in SEQ ID NOS: 57 and 58, respectively. The resulting 321 basepair PCR product was ligated into pTTVH8G immediately downstream of thesignal peptide sequence of human VEGF A (NM_003376). This cloning stepalso positioned unique restriction endonuclease sites that permitted theprecise positioning of the cDNAs encoding the mouse light chain variableregions. The sequence of the final expression plasmid, called pTTVK1, isshown in SEQ ID NO.:59. Based on the sequences disclosed in Table 2, PCRprimers specific for the light chain variable regions of antibodies 3D3,3G10, 3C4 and 3A4 (SEQ ID NOS:15, 19, 23 and 47, respectively) weredesigned that incorporated, at their 5′-end, a sequence identical to thelast 20 base pairs of the VEGF A signal peptide. The sequences of theseprimers are shown in SEQ ID NOS:60, 61, 62 and 213. The same reverseprimer was used to amplify all three light chain variable regions of3D3, 3G10 and 3C4 since the extreme 3′-ends were identical. This primer(SEQ ID NO.:63) incorporated, at its 3′-end, a sequence identical to thefirst 20 base pairs of the human kappa constant domain. Primer SE IDNO.:214 was used to amplify the 3A4 light chain variable region. Boththe PCR fragments and the digested pTTVK1 were treated with the 3′-5′exonuclease activity of T4 DNA polymerase resulting in complimentaryends that were joined by annealing. The annealing reactions weretransformed into competent E. coli and the expression plasmids wereverified by sequencing to ensure that the mouse light chain variableregions were properly inserted into the pTTVK1 expression vector. Thoseskilled in the art will readily recognize that the method used forconstruction of the light chain expression plasmids applies to allanti-KAAG1 antibodies contained in the original Fab library.

Heavy chain expression vector—the expression vector that produced theheavy chain immunoglobulins was designed in a similar manner to thepTTVK1 described above for production of the light chainimmunoglobulins. Plasmid pYD11 (Durocher et al., 2002), which containsthe human IgGK signal peptide sequence as well as the CH2 and CH3regions of the human Fc domain of IgG1, was modified by ligating thecDNA sequence encoding the human constant CH1 region. PCR primersOGS1769 and OGS1770 (SEQ ID NOS:64 and 65), designed to contain uniquerestriction endonuclease sites, were used to amplify the human IgG1 CH1region containing the nucleotide sequence and corresponding amino acidsequence shown in SEQ ID NOS:66 and 67. Following ligation of the 309base pair fragment of human CH1 immediately downstream of the IgGKsignal peptide sequence, the modified plasmid (SEQ ID NO.:68) wasdesignated pYD15. When a selected heavy chain variable region is ligatedinto this vector, the resulting plasmid encodes a full IgG1 heavy chainimmunoglobulin with human constant regions. Based on the sequencesdisclosed in Table 2, PCR primers specific for the heavy chain variableregions of antibodies 3D3, 3G10, 3C4 and 3A4 (SEQ ID NOS:17, 21, 25 and45, respectively) were designed that incorporated, at their 5′-end, asequence identical to the last 20 base pairs of the IgGK signal peptide.The sequences of these primers are shown in SEQ ID NOS:69 (3D3 and 3G10have the same 5′-end sequence), SEQ ID NO.: 70 or SEQ ID NO.:215 for3A4. The same reverse primer was used to amplify all three heavy chainvariable regions of 3D3, 3C4 and 3G10 since the extreme 3′-ends wereidentical. This primer (SEQ ID NO.:71) incorporated, at its 3′-end, asequence identical to the first 20 base pairs of the human CH1 constantdomain. For the 3A4 heavy chain variable region, SEQ ID NO.:216 wasused. Both the PCR fragments and the digested pYD15 were treated withthe 3′-5′ exonuclease activity of T4 DNA polymerase resulting incomplimentary ends that were joined by annealing. The annealingreactions were transformed into competent E. coli and the expressionplasmids were verified by sequencing to ensure that the mouse heavychain variable regions were properly inserted into the pYD15 expressionvector. Those skilled in the art will readily recognize that the methodused for construction of the heavy chain expression plasmids applies toall anti-KAAG1 antibodies contained in the original Fab library.

Expression of human IgG1s in 293E cells—The expression vectors preparedabove that encoded the light and heavy chain immunoglobulins wereexpressed in 293E cells using the transient transfection system(Durocher et al., 2002). Other methods of transient or stable expressionmay be used. The ratio of light to heavy chain was optimized in order toachieve the most yield of antibody in the tissue culture medium and itwas found to be 9:1 (L:H). The ability of the anti-KAAG1 antibodies(monoclonal, chimeric or humanized) to bind to recombinant Fc-KAAG1 wasmeasured by ELISA and compared with the original mouse Fabs.

The scheme used to convert other Fabs into a complete IgG (including the3A4) and for expression of the antibodies is described in more detailsin international application No. PCT/CA2012/000296, the entire contentof which is incorporated herein by reference.

Example 2 Humanization of the 3A4 Mouse Monoclonal Antibody

International patents No. PCT/CA2009/001586, PCT/CA2010/001795 and No.PCT/CA2012/000296, described exemplary methodology used to generate thehumanized light chain and heavy chain variable regions.

Humanization of the 3A4 antibody light chain variable region involved 11mutations to its proposed humanized framework for 100% frameworkhumanization. Humanization of the 3A4 antibody heavy chain variableregion involved 23 mutations to its proposed humanized framework for100% framework humanization. These 100% humanized variable regionsequences are labelled Lvh1 and Hvh1, respectively (SEQ ID NOs:189 and194). Additional humanized sequences were also designed in which severalresidues from the 3A4 mouse sequences were retained based on carefulstructural and comparative sequence analyses that indicate a highprobability of altering antigen-binding affinity if mutations are to beintroduced at these positions. These sequences of the variable regionsare labelled Lvh2, Hvh2, Hvh3 and Hvh4 (SEQ ID NOs: 190, 195, 196 and197).

The two humanized light chain variants (including the constant region)are identified herein as Lh1 (SEQ ID NO.: 199) and Lh2 (SEQ ID NO.:200).The four humanized heavy chain variants (including the constant regionare identified herein as Hh1 (SEQ ID NO.:202), Hh2 (SEQ ID NO.:203), Hh3(SEQ ID NO.:204) and Hh4 (SEQ ID NO.:205). The two humanized light chainand 4 humanized heavy chain can be assembled into 8 humanized antibodies(Lh1Hh1, Lh1Hh2, Lh1Hh3, Lh1Hh4, Lh2Hh1, Lh2Hh2, Lh2Hh3, and Lh2Hh4).

In the case of 3A4 light-chain humanized sequence Lvh2 (SEQ ID NO:190),framework residues Val-L2 and Lys-L45 were retained from the mousesequence since residue L2 is semi-buried, contacts both CDR-L1 andCDR-L3, and has antigen-contacting propensity, while residue L45approaches the heavy-chain. We note that both these murine residues mayoccur in human frameworks. In the case of 3A4 heavy-chain humanizedsequence Hvh2 (SEQ ID NO:195), framework residues Ile-H2 and Lys-L73were retained from the mouse sequence since residue H2 is semi-buried,contacts both CDR-H1 and CDR-H3, and has antigen-contacting propensity,while residue H73 belongs to the Vernier zone supporting CDR-H2, andboth these murine residues may occur in human frameworks. In the case of3A4 heavy-chain humanized sequence Hvh3 (SEQ ID NO:196), Ile-H2 andLys-L73 back-mutations were retained and in addition to these, frameworkresidues Ile-H48, Ala-H67, Leu-H69 and Val-H71 were retained from themouse sequence since all these additional murine residues are buriedresidues and belong to the Vernier zone supporting CDR-H2, and alsomurine residue H71 may occur in human frameworks. In the case of 3A4heavy-chain humanized sequence Hvh4 (SEQ ID NO:197), all 6back-mutations of the Hvh3 humanized variant were included plusadditional two mouse framework residues Lys-H38 and Lys-H66 since theyrepresent semi-buried residues close to CDR-H2. The resulting amino acidsequences of the murine and humanized chains are listed in Table 1. Thealignment of the murine and humanized light chain variable regions isshown in FIG. 1a and the alignment of the murine and humanized heavychain variable regions is shown in FIG. 1 b.

FIGS. 2a and 2b is an alignment of the murine light chain variableregion with the 100% humanized light chain variable region and themurine heavy chain variable region with the 100% humanized heavy chainvariable region respectively. This figure illustrates the amino acidsthat are preserved and those that have been chosen for substitution.

Example 3 Assembly and Expression of 3A4 Humanized Variant Antibodies

The purpose of these investigations is to determine the kineticsparameters of anti-clusterin antibodies. In particular, to determinewhether the humanization of the 3A4 anti-KAAG1 monoclonal antibodyaffects the kinetics parameters of its binding to human KAAG1. To thisend, a kinetic analysis method was developed using the ProteOn XPR36instrument from BioRad. Human KAAG1 was immobilized on a sensor chip.Full length antibodies or Fab fragments were injected and allowed tointeract with the immobilized KAAG1.

Construction of Plasmid Encoding the Chimeric (Murine) Heavy and LightChains of 3A4

The heavy and light chains of the chimeric antibody were amplified byPCR from the original murine immunoglobulin chains using the followingoligonucleotide primer pairs: heavy chain, 5′-oligo encoded by SEQ IDNO: 206 and 3′-oligo encoded by SEQ ID NO:207; light chain, 5′-oligoencoded by SEQ ID NO: 208 and 3′-oligo encoded by SEQ ID NO:209. Theresulting PCR products were digested by Hind III and cloned into pK-CR5(SEQ ID NO:210) previously digested with Hind III.

Construction of Plasmids Encoding the Humanized Heavy Chain 3A4 Variants1, 2, 3 and 4

The fragments coding for the humanized heavy chain region of theantibody 3A4 (Hh1, Hh2, Hh3 and Hh4) were ordered from GenScript(Piscataway, USA). The DNA fragments including the kozak and stop codonsequences were digested with HindIII and cloned into the HindIII site ofplasmid pK-CR5 previously dephosphorylated with calf intestinalphosphatase (NEB) to prevent recircularization. FIG. 3a shows the map ofthe plasmid pK-CR5-3A4-HC-variant1. All heavy chain variants of thehumanized 3A4 were constructed in a similar manner.

Construction of Plasmids Encoding the Humanized Light Chain 3A4 Variants1 and 2

The fragments coding for the human light chain regions of the antibody3A4 (Lh1 and Lh2) were ordered from GenScript. The DNA fragmentsincluding the kozak and stop codon sequences was digested with BamHI andcloned into the BamHI site of plasmid pMPG-CR5 (SEQ ID NO:211)previously dephosphorylated with calf intestinal phosphatase (NEB) toprevent recircularization. FIG. 3b shows the map of the plasmidpMPG-CR5-3A4-LC-variant1. All light chain variants of the humanized 3A4were constructed in a similar manner.

Transient Transfection Study

Plasmid DNA was isolated from small cultures of E. coli using theMini-Prep kit (Qiagen Inc, Mississauga, ON) according to themanufacturer's recommendation. Briefly, 2 ml of LB medium containing 100μg/ml of ampicillin were inoculated with a single colony picked afterligation and transformation. The cultures were incubated at 37° C.overnight with vigorous shaking (250 RPM). The plasmid was then isolatedfrom 1.5 ml of culture using the protocols, buffers, and columnsprovided by the kit. The DNA was eluted using 50 μl of sterile water.Plasmid DNA was isolated from large culture of E. coli using the PlasmidPlus Maxi kit (Qiagen Inc, Mississauga, ON) according to themanufacturer's recommendation. 200 mL of LB medium containing 100 μg/mLampicillin were inoculated with a single fresh colony of E. coli andincubated overnight at 37° C. with vigorous shaking (250 RPM). Thebacteria (130 mL of culture for the heavy chain and 180 mL of culturefor the light chain) were pelleted by centrifugation at 6000×g, for 15min, at 4° C. and the plasmid was isolated using the protocols, buffersand columns provided by the kit. The pure plasmids was resuspended insterile 50 mM Tris, pH8 and quantified by measuring the optical densityat 260 nm. Before transfection the purified plasmid were sterilized byextraction with phenol/chloroform followed by ethanol precipitation. Theplasmid were resuspended in sterile 50 mM Tris, pH 8 and quantified byoptical density at 260 nm.

Before transfection, the cells (CHO-cTA) were washed with PBS andresuspended at a concentration of 4.0×10⁶ cell/ml in growth medium(CD-CHO, Invitrogen) without dextran sulfate for 3 h in suspensionculture. For each plasmid combination, 45 ml of cells were transfectedby adding slowly 5 ml of CDCHO medium supplemented with 10 μg/ml of eachplasmid and 50 μg/ml of polyethylenimine (PEI Max; Polysciences). Thefinal concentration was 1 μg/ml of each plasmid and 5 μg/ml of PEI.After 2 h, the cells were transferred at 30° C. The next days, 50 μg/mLof dextran sulfate and 3.75 ml of each supplement (Efficient Feed A andB Invitrogen) were added to the cells and they were incubated at 30° C.for 13 days. 2.5 ml of Feed A and 2.5 ml of Feed B were added at day 4,6, 8 and 11. On day 13, the supernatant was clarified by centrifugationand filtered through a 0.22 μM filter.

CHO cells (CHOcTA) were transfected with plasmids encoding the differentvariants of humanized heavy and light chains of the 3A4 antibodyregulated by the CR5 promoter. Transfection with different combinationsof light and heavy chains was performed. As control, cells were alsotransfected with plasmids encoding the chimeric/murine antibody.

Purification of Antibody

15 ml of supernatant from the CHO cell transfections were concentratedby centrifugation using the Amicon Ultra (Ultacell-50k) cassette at 1500rpm. The concentrated antibody (550 μl) was purified using the Nab spinkit Protein A Plus (Thermo Scientific) according to the manufacture'srecommendations. The purified antibodies were then desalted using PBSand the concentrating Amicon Ultra (Ultracel-10K) cassette at 2500 rpmto a final volume of 250 μl. The purified antibody was quantified byreading the OD₂₈₀ using the Nanodrop spectrophotometer and kept frozenat −20° C. An aliquote of the purified antibody was resuspended into anequal volume of Laemmli 2× and heated at 95° C. for 5 min and chilled onice. A standard curve was made using known amount of purified human IgG1kappa from Human Myeloma plasma (Athens Research). The samples wereseparated on a polyacrylamide Novex 10% Tris-Glycine gel (InvitrogenCanada Inc., Burlington, ON) and transferred onto a Hybond-Nnitrocellulose membrane (Amersham Bioscience Corp., Baie d'Urfée, QC)for 1 h at 275 mA. The membrane was blocked for 1 h in 0.15% Tween 20,5% skimmed milk in PBS and incubated for 1 hr with an Goat anti-HumanIgG (H+L) conjugated to Cy5 (Jackson, Cat#109-176-099). The signal wasrevealed and quantified by scanning with the Typhoon Trio+ scanner (GEHealthcare). As shown in FIG. 4, all combinations of the 3A4 humanizedantibody variants were expressed in CHO cells.

Example 4 Kinetic Analysis of Murine and Humanized 3A4 Antibody Supplies

GLM sensorchips, the Biorad ProteOn amine coupling kit (EDC, sNHS andethanolamine), and 10 mM sodium acetate buffers were purchased fromBio-Rad Laboratories (Mississauga, ON). HEPES buffer, EDTA, and NaClwere purchased from from Sigma-Aldrich (Oakville, ON). Ten percent Tween20 solution was purchased from Teknova (Hollister, Calif.). The goatanti-human IgG Fc fragment specific antibody was purchased from JacksonImmunoResearch. The gel filtration column Superdex 75 10/300 GL waspurchased from GE Healthcare.

Gel Filtration

The KAAG1 protein at a concentration of 3.114 mg/ml and a volume of 220μL was injected onto the Superdex G75 column. The separation was done at0.4 ml/min in HBST running buffer (see below) without Tween 20. Thevolume of the fractions collected was 500 μL. Concentration of KAAG1 ineach fraction was determined by OD₂₈₀ using an extension coefficient of5500 and a MW of 8969. FIG. 5 represents the profile of the gelfiltration of KAAG1. A small peak of potential aggregate is eluting ataround 11 ml. The protein eluting at 13 ml was used as analyte for theSPR assay (fractions 15-19).

SPR Biosensor Assays

All surface plasmon resonance assays were carried out using a BioRadProteOn XPR36 instrument (Bio-Rad Laboratories Ltd. (Mississauga, ON)with HBST running buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and0.05% Tween 20 pH 7.4) at a temperature of 25° C. The anti-mouse Fccapture surface was generated using a GLM sensorchip activated by a 1:5dilution of the standard BioRad sNHS/EDC solutions injected for 300 s at30 μL/min in the analyte (horizontal) direction. Immediately after theactivation, a 13 μg/mL solution of anti-human IgG Fc fragment specificin 10 mM NaOAc pH 4.5 was injected in the analyte direction at a flowrate of 25 μL/min until approximately 8000 resonance units (RUs) wereimmobilized. Remaining active groups were quenched by a 300 s injectionof 1M ethanolamine at 30 μL/min in the analyte direction, and this alsoensures mock-activated interspots are created for blank referencing. Thescreening of the 3A4 variants for binding to KAAG1 occurred in twosteps: an indirect capture of 3A4 variants from cell supernatant ontothe anti-human IgG Fc fragment specific surface in the ligand direction(vertical) followed by a KAAG1 injection in the analyte direction.Firstly, one buffer injection for 30 s at 100 uL/min in the liganddirection was used to stabilize the baseline. For each 3A4 capture,unpurified 3A4 variants in cell-culture media were diluted to 4% inHBST, or approximately 1.25 μg/mL of purifed 3A4 in HBST was used. Fourto five 3A4 variants along with wild-type 3A4 were simultaneouslyinjected in individual ligand channels for 240 s at flow 25 μL/min. Thisresulted in a saturating 3A4 capture of approximately 400-700 RUs ontothe anti-human IgG Fc fragment specific surface. The first ligandchannel was left empty to use as a blank control if required. This 3A4capture step was immediately followed by two buffer injections in theanalyte direction to stabilize the baseline, and then the gel filtrationpurified KAAG1 was injected. For a typical screen, five KAAG1concentrations (8, 2.66, 0.89, 0.29, and 0.098 nM) and buffer controlwere simultaneously injected in individual analyte channels at 50 μL/minfor 120 s with a 600s dissociation phase, resulting in a set of bindingsensorgrams with a buffer reference for each of the captured 3A4variants. The anti-human IgG Fc fragment specific-3A4 complexes wereregenerated by a 18 s pulse of 0.85% phosphoric acid for 18 s at 100μL/min to prepare the anti-human IgG Fc fragment specific surface forthe next injection cycle. Sensorgrams were aligned and double-referencedusing the buffer blank injection and interspots, and the resultingsensorgrams were analyzed using ProteOn Manager software v3.0. Thekinetic and affinity values were determined by fitting the referencedsensorgrams to the 1:1 Langmuir binding model using local R_(max), andaffinity constants (K_(D) M) were derived from the resulting rateconstants (k_(d) s⁻¹/k_(a) M⁻¹s⁻¹).

Determination of Rate and Affinity Constants

FIG. 6 summarizes the association (k_(a), 1/Ms) and dissociation (k_(d),1/s) rate constants as well as affinity (K_(D), M) constants for theinteraction of KAAG1 with purified murine 3A4, murine 3A4 transientlyexpressed as a chimeric and transiently expressed humanized variants.These constants are graphically represented in FIG. 7a-c . Theassociation rate constant is very similar for the pure parental,chimeric and humanized 3A4 variants (FIG. 7a ). The dissociation rateconstants is similar for the transiently express chimeric as compared tothe pure parental 3A4 with suggest that the transfection procedure didnot alter the parameters of the interaction of KAAG1 with the antibody(FIG. 7b ). However, all humanized variants seem to have a slightlyaltered off rate, i.e. quicker dissociation rate (FIG. 7b ). This isreflected in the affinity constants (FIG. 7c ). In summary, there is alinear correlation between the binding affinity (log K_(D)) of thehumanized variant and the number of back-mutations made in the parentantibody (LcHc) with a decrease in the binding affinity as the number ofmutations is increasing. However, the difference in binding affinity isonly 4 fold different between the worse variant (H1L1, 0.47 nM) whichhas no mouse residue retained and the best variant which has 10 mouseresidues retained (H4L2, 0.1 nM). Finally, the binding affinity of allvariants for KAAG1 was found to be sub-nanomolar and the best variant(H4L2, 0.1 nM) exhibited an affinity about 6-fold weaker than the murine(LcHc, 0.057 nM). Overall, these results indicate that humanization wassuccessful as all of the variants displayed high affinity for KAAG1.

Example 5 Binding of 3A4 Humanized Variants to KAAG1 in an ELISA

ELISA methods were also used to compare the binding activity of thehumanized 3A4 variants to the murine 3A4 antibody. Recombinant humanKAAG1 was coated in 96-well plates O/N, washed and incubated for 1 h atRT with increasing quantities of murine or humanized 3A4 variants.Following another round of washing steps, an anti-human antibodyconjugated to HRP was added to the wells and the bound 3A4 antibody wasmeasured calorimetrically at Abs₄₅₀. As shown in FIG. 8a , the humanizedvariants (Lh1Hh1, Lh1Hh2, Lh1Hh3 and Lh1Hh4) displayed very similarbinding to KAAG1 when compared to the murine 3A4 (LcHc), which has ahigh affinity of 0.016 nM. This result indicated that all four humanizedheavy chain variants were comparable to the original h3A4 heavy chainwhen assembled with the L1 variant of the humanized light chain. FIG. 8ashows the results when the heavy chain variants were assembled with Lh2variant of the 3A4 humanized light chain. In this instance, there was adifference in the binding of the variants. For example, Lh2hh4 was thevariant with the closest profile compared to the murine 3A4. This was inagreement with the SPR data, which showed that the variant 4 of theheavy chain had the highest affinity for KAAG1. Taken together, thesebinding results show that the humanized variants all interact with humanKAAG1 in this assay. Although there were some subtle differences, thebinding in ELISA was in concordance with the SPR results.

Example 6 Binding of 3A4 Humanized Variants on the Surface of CancerCells

Flow cytometry was used to evaluate the capacity of the humanized 3A4variants to interact with KAAG1 expressed on the surface of cancercells. To this end, SKOV-3 ovarian cancer cells, which we had previouslyshowed were efficiently bound by 3A4 by flow cytometry, were incubatedwith the eight humanized variants and the original murine antibody.Briefly, SKOV-3 cells were detached from the plate with EDTA andincubated on ice with either 3.0 mg/ml, 0.3 mg/ml or 0.3 mg/ml of theantibodies for 1 h. After three washing steps, the cells were incubatedwith the secondary antibody, anti-human IgG-conjugated to FITC for 1 hon ice. Cell surface fluorescence was measured in a flow cytometer andthe values are shown in the histogram of FIG. 9. As depicted, allvariants could detect KAAG1 on the surface on unpermeabilized and thestrongest signals were obtained at the highest concentration of 3A4antibodies (3 mg/ml) and decreased as the concentration of the antibodywas decreased. Among the different variants, the ones with the mostmurine back-mutations (FIG. 9, see Lh1Hh4 and Lh2Hh4) interacted withKAAG1 on the surface of cells with the highest activity. In fact, Lh1Hh4and Lh2hh4 appeared to be slight improved cell surface binding to KAAG1compared to the murine 3A4 antibody (LcHc).

Example 7

This example describes the use of anti-KAAG1 antibodies for detectingthe expression of KAAG1 in TNBC.

As a means of determining if the KAAG1 antigen was present in TNBCsamples, immunohistochemistry was conducted. Tissue microarrays wereobtained that contained 139 breast tumor samples generated from patientbiopsies. Paraffin-embedded epithelial breast tumor samples were placedon glass slides and fixed for 15 min at 50° C. Deparaffinization wasconducted by treating 2× with xylene followed by dehydration insuccessive 5 min washes in 100%, 80%, and 70% ethanol. The slides werewashed 3× in PBS for 5 min and treated with antigen retrieval solution(1 mM EDTA, pH 8.0) to unmask the antigen. Endogenous peroxide reactivespecies were removed by incubating slides with H₂O₂ in methanol andblocking was performed by incubating the slides with serum-free blockingsolution (Santa Cruz Biotech) for 5 min at room temperature. The primaryantibody (anti-KAAG1 3A4) was added for 1 h at room temperature.KAAG1-reactive antigen was detected by incubating with biotin-conjugatedmouse anti-kappa followed by streptavidin-HRP tertiary antibody.Positive staining was revealed by treating the slides with DAB-hydrogenperoxide substrate for less than 5 min and subsequently counterstainedwith hematoxylin. The KAAG1 protein was found to be expressed at veryhigh levels in the vast majority of breast tumor samples. Arepresentative array containing 139 tumors is depicted in FIG. 10. Inparticular, 15/20 biopsy samples confirmed to be TNBC (FIG. 10, samplesidentified by an asterisk) were stained strongly for KAAG1 expressionwith the 3A4 antibody. Taken together, these immunohistochemical studiesillustrate the utility of detecting KAAG1 in breast cancer, inparticular TNBC, with the monoclonal antibodies.

Example 8

This example describes the use of anti-KAAG1 antibodies for detectingthe expression of KAAG1 in TNBC cell lines.

Combined results from the bioinformatics analysis of the primarystructure of the cDNA encoding KAAG1, biochemical studies, andimmunohistochemical detection of the protein in epithelial cellssuggested that the KAAG1 antigen was located at the cell surface.However, more direct evidence was required to demonstrate that KAAG1 isindeed expressed on the surface of TNBC cells. To conduct this analysis,breast cancer cell lines were obtained from a commercial vendor (ATCC,Manassas, Va.) and used in flow cytometry experiments. RT-PCR expressionanalyses using KAAG1 mRNA specific primers previously showed thatcertain breast cancer cell lines expressed KAAG1 mRNA (seePCT/CA2007/001134). Therefore some of these cell lines were selected todetermine the presence of the KAAG1 antigen at their surface. To verifythis, the triple-negative MDA-MB-231, MDA-MB-436, MDA-MB-468, BT-20 andBT-549 cell lines were tested for surface expression of KAAG1 using the3A4 anti-KAAG1 antibody. In addition, breast cancer cell lines, whichare not triple-negative, namely T47D and MCF-7, were also included inthe analysis. Finally, a control cell line, 293-6E, that exhibitsundetectable level of KAAG1 antigen expression was included as anegative control for the flow cytometry experiment (FCM). For thepurpose of FCM analysis, the cells were harvested using 5 mM EDTA,counted with a hemocytometer, and resuspended in FCM buffer (0.5% BSA,0.01% goat serum in 1×PBS) at a cell density of 2×10⁶ cells/ml. Chimeric3A4 anti-KAAG1 antibody or a control IgG were added to 100 μl of cellsat a final concentration of 0.5 μg/ml and incubated on ice for 1 h. Thecells were washed in cold FCM buffer to remove unbound antibodies,resuspended in 100 μl FCM buffer containing anti-human IgG conjugated toFITC secondary antibody (diluted 1:200) and incubated on ice for 45 min.Following another washing step in cold FCM buffer, the cells wereresuspended in 300 μl FCM buffer and analyzed with a flow cytometer. 10μg/ml propidium iodide was added to each sample to allow for gating ofdead cells. The results from three independent experiments are shown inFIG. 11, where the mean fluorescence intensity (MFI) fold Inductionrepresents the geometric mean value of the signal obtained when thecells were incubated with 3A4 antibody over that of the negative humanIgG control, which was arbitrarily set to 1. Incubation of theantibodies with the control 293-6EHEK-293 cells resulted in fluorescencesignals that were similar to the signal obtained when the cells wereincubated in the absence of the primary antibody. Furthermore, there wasno significant difference between the signal obtained with 3A4 comparedto the control IgG. Moreover, when the control IgG was incubated withthe breast cancer cell lines, the signals were very similar to thoseobtained with the control 293-6E cells. By contrast, detectablefluorescence signal was observed when the 3A4 antibody was incubatedwith all breast cancer cells lines. Although variable amount offluorescence was observed, the highest amount of KAAG1 was detected onthe surface of MDA-MB-231 and BT-20 cell lines, two TNBC cell lines (seeFIG. 11, TNBC cell lines are indicated with an asterisk). In fact allfive TNBC cell lines were positive for KAAG1 expression under theseconditions. T47 D and MCF-7 cells also expressed KAAG1. Taken together,this flow cytometry analysis shows that TNBC cell line express highlevel of KAAG1 on their cell surface.

Example 9

Methods for Use of the 3A4 Anti-KAAG1 Antibody as an Antibody Conjugate

As demonstrated above, the KAAG1 antigen was detected by 3A4 on thesurface of cancer cells using flow cytometry. There are severaldifferent molecular events that can occur upon binding of an antibody toits target on the surface of cells. These include i) blockingaccessibility to another cell-surface antigen/receptor or a ligand, ii)formation of a relatively stable antibody-antigen complex to allow cellsto be targeted via ADCC or CDC, iii) signalling events can occur asexemplified by agonistic antibodies, iv) the complex can beinternalized, or v) the complex can be shed from the cell surface. Toaddress this question we examined the behavior of the 3A4 antibody-KAAG1complex on the surface of the cells. The ovarian cancer cell line,SKOV3, was used as a positive control in this experiment since it wassuccessfully used in previous internalization experiments (seePCT/CA2009/001586). MDA-MB-231 TNBC cells were plated, washed, andincubated with 0.5 μg/ml chimeric 3A4 antibody as described in Example3. After washing, complete medium was added and the cells placed at 37°C. for up to 60 minutes. The cells were removed at the indicated times(see FIG. 12), rapidly cooled, prepared for flow cytometry withFITC-conjugated anti-human IgG and the results were expressed as thepercentage of mean fluorescence intensity remaining on the cell surfacecompared with the signal at time 0 minutes (see FIG. 12, Surface signal(% remaining at 0 min). As illustrated in FIG. 12, the fluorescencesignal decreased rapidly when 3A4 was incubated with MDA-MB-231 cells(FIG. 12, black bars, indicated by MDA-231 in the figure) and seemed toachieve a maximum loss of signal by 30-45 minutes. The loss of signalwas comparable to that observed when 3A4 was incubated with the SKOV3cells (FIG. 12, grey bars). This result indicates that the 3A4/KAAG1complex disappeared from the cells which indicated that aninternalization of the complex likely occurred. Preliminary studies toelucidate the mechanism responsible for this decrease in cell-surfacefluorescence have revealed that the complex appears to be internalized.Similar results are expected with humanized 3A4 antibodies.

Similar results were observed in two additional TNBC cell lines, namelyMDA-MB-436 (FIG. 13) and BT-20 (FIG. 14) confirming that theinternalization of the 3A4/KAAG1 complex on the surface of multiple TNBCcell lines. By contrast, despite similar MFI levels of 3A4 binding onthe surface of MDA-MB-436 and T47D (FIG. 11), the loss of signal at thecell surface was not observed when 3A4 was incubated with the T47D cellline. This finding suggests the possibility that internalization of the3A4/KAAG1 complex might occur to a higher degree in TNBC cells (FIG. 15)compared with cells that are not triple-negative.

These findings were further confirmed by conducting immunofluorescenceon live cells to see if this internalization could be microscopicallyobserved. MDA-MB-231 cells were seeded on cover slips and once the cellswere properly adhered, fresh medium was added containing the 3A4anti-KAAG1 chimeric antibody at 10 ug/ml and incubating at 37 C for 4 h.The cells were washed in PBS then fixed in 4% paraformaldehyde (in PBS)for 20 min. After washing, the cells were permeabilized with 0.1% TritonX-100 in PBS for 5 min. Blocking was performed with 1.5% dry milk in PBSfor 1 h. Lysosomal-associated membrane protein 1 (LAMP1, Chang et al.,2002) was detected by incubating with anti-LAMP1 (Santa Cruz, sc-18821,diluted 1:100) in 1.5% milk in PBS for 2 h. After washing in PBS, thesecondary antibodies were added together in 1.5% milk and incubated for1 h. For the anti-KAAG1 chimeric antibody the secondary antibody was aRhodamine Red-X conjugated donkey anti-human IgG (H+L) diluted 1:300.For the anti-LAMP1 antibody the secondary antibody was aDyLight488-conjugated goat anti-mouse IgG (H+L) diluted 1:300. Bothsecondary antibodies were from Jackson ImmunoResearch. The coverslipswere washed in PBS and mounted in ProLong Gold antifade reagent withDAPI. As seen in FIG. 7, after 4 hours of incubation at 37 C in thepresence of MDA-MB-231 cancer cells, the 3A4 antibody was able to bedetected in complexes predominantly near the peri-nuclear area (arrows,see red staining in the left panel in FIG. 16), which is typical ofendosomal-lysosomal-based internalization pathways. This observation wasfurther confirmed when a lysosomal marker, LAMP1 was visualized and wasfound to be also expressed in these areas (arrows, see green staining inthe middle panel in FIG. 16). Importantly, the merging of the two imagesresulted in the appearance of yellow-orange structures indicating thatthe 3A4 and the anti-LAMP1 antibodies were present in the samestructures (arrows, see yellow staining in the right panel in FIG. 16).The co-localization of 3A4, which binds to KAAG1 on the surface ofcancer cells, with LAMP1, a marker of late endosomes/lysosomes, showsthat the antibody/antigen complex was internalized and that it follows apathway that is amenable for the release of a payload that would beconjugated to the 3A4 antibody. Identical results were observed inanother TNBC cell line, BT-20 (see FIG. 17).

Taken together, these studies demonstrated that antibodies specific forKAAG1 such as 3A4 might have uses as an antibody conjugate, inparticular, as an antibody-drug conjugate (ADC). Thus, the high level ofTNBC specificity of KAAG1 coupled with the capacity of this target to beinternalized in cells support the development of applications as an ADC.

Example 10

In order to demonstrate that anti-KAAG1 antibodies can efficientlytarget and kill cells lacking ER protein expression, PgR proteinexpression and/or showing absence of HER2 protein over-expression, wegenerated two antibody drug conjugates (ADCs); 3A4-ADC1 and 3A4-ADC2.

To that effect, we used the chimeric 3A4 antibody and conjugated acytotoxic drug via a highly stable peptide linker that is selectivelycleaved by lysosomal enzymes after internalization (3A4-ADC1), orconjugated with another anti-mitotic drug via a non-cleavable linker(3A4-ADC2). The cytotoxic drug may become active once internalized inthe cells.

The ability of the 3A4 ADCs to detect KAAG1 on the surface of TNBC cellswas determined using flow cytometry using the methods described herein.Briefly, unconjugated 3A4, 3A4-ADC1, 3A4-ADC2 and a control IgG wereincubated in the presence of MDA-231 TNBC cells, which are KAAG1positive. Results indicated that the conjugation of 3A4 with either drugdid not affect its binding to triple negative breast cancer cells suchas MDA-231 (data not shown).

Having confirmed that the 3A4 ADCs could bind to KAAG1 expressed on thesurface of TNBC cells, their cytotoxicity against these cells wasevaluated in cell proliferation assays. MDA-231 or TOV-112D cells werecultured as described above in previous examples. The cells were seededat 3000 cells/well in 96-well plates in 200 μl of media per wellovernight at 37° C., in 5% CO₂. The next day, media was replaced withfresh media containing antibodies, at concentrations ranging from 0.122nM to 500 nM, and incubated at 37° C. for 72 h. All conditions wereperformed in triplicate wells. The number of surviving cells wasdetermined by performing a cellular proliferation assay, using CellTiter96 Aqueous One Solution (Promega, Madison, Wis.), followingmanufacturer's protocol. Following the collection of the raw data, theresults were expressed as the percentage survival compared to the numberof cells in the wells treated with PBS, which was set to 100%. Resultsindicated that the unconjugated 3A4 did not affect the proliferation ofMDA-231 cells at all concentrations tested. In contrast, the 3A4 ADCstested showed significant cytotoxicity.

These results indicate that 3A4 antibody conjugates may be used as analternative treatment for patients having triple negative breast canceror basal-like breast cancer. Similar results are expected for conjugatesbased on humanized 3A4 antibodies.

The present description refers to a number of documents, the content ofwhich is incorporated herein by reference in their entirety.

Sequences Referred to in the Description

SEQ ID NO.: 1GAGGGGCATCAATCACACCGAGAAGTCACAGCCCCTCAACCACTGAGGTGTGGGGGGGTAGGGATCTGCATTTCTTCATATCAACCCCACACTATAGGGCACCTAAATGGGTGGGCGGTGGGGGAGACCGACTCACTTGAGTTTCTTGAAGGCTTCCTGGCCTCCAGCCACGTAATTGCCCCCGCTCTGGATCTGGTCTAGCTTCCGGATTCGGTGGCCAGTCCGCGGGGTGTAGATGTTCCTGACGGCCCCAAAGGGTGCCTGAACGCCGCCGGTCACCTCCTTCAGGAAGACTTCGAAGCTGGACACCTTCTTCTCATGGATGACGACGCGGCGCCCCGCGTAGAAGGGGTCCCCGTTGCGGTACACAAGCACGCTCTTCACGACGGGCTGAGACAGGTGGCTGGACCTGGCGCTGCTGCCGCTCATCTTCCCCGCTGGCCGCCGCCTCAGCTCGCTGCTTCGCGTCGGGAGGCACCTCCGCTGTCCCAGCGGCCTCACCGCACCCAGGGCGCGGGATCGCCTCCTGAAACGAACGAGAAACTGACGAATCCACAGGTGAAAGAGAAGTAACGGCCGTGCGCCTAGGCGTCCACCCAGAGGAGACACTAGGAGCTTGCAGGACTCGGAGTAGACGCTCAAGTTTTTCACCGTGGCGTGCACAGCCAATCAGGACCCGCAGTGCGCGCACCACACCAGGTTCACCTGCTACGGGCAGAATCAAGGTGGACAGCTTCTGAGCAGGAGCCGGAAACGCGCGGGGCCTTCAAACAGGCACGCCTAGTGAGGGCAGGAGAGAGGAGGACGCACACACACACACACACACAAATATGGTGAAACCCAATTTCTTACATCATATCTGTGCTACCCTTTCCAAACAGCCTA SEQ ID NO.: 2MDDDAAPRVEGVPVAVHKHALHDGLRQVAGPGAAAAHLPRWPPPQLAASRREAPPLSQRPHRTQGAGSPPETNEKLTNPQVKEK SEQ ID NO.: 3GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAATAGGACAGAAGGTCACTATGAACTGCAAGTCCAGTCAGAGCCTTTTAAATAGTAACTTTCAAAAGAACTTTTTGGCCTGGTACCAGCAGAAACCAGGCCAGTCTCCTAAACTTCTGATATACTTTGCATCCACTCGGGAATCTAGTATCCCTGATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGATTACTTCTGTCAGCAACATTATAGCACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGCTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT SEQ ID NO.: 4DIVMTQSPSSLAVSIGQKVTMNCKSSQSLLNSNFQKNFLAWYQQKPGQSPKLLIYFASTRESSIPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELKAVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.: 5GAGGTTCAGCTGCAGCAGTCTGTAGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACATATTTACTGACTATGAGATACACTGGGTGAAGCAGACTCCTGTGCATGGCCTGGAATGGATTGGGGTTATTGATCCTGAAACTGGTAATACTGCCTTCAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACATATCCTCCAGCACAGCCTACATGGAACTCAGCAGTTTGACATCTGAGGACTCTGCCGTCTATTACTGTATGGGTTATTCTGATTATTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCTCAACGAAGGGCCCATCTGTCTTTCCCCTGGCCCCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGAATTCACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAA SEQ ID NO.: 6EVQLQQSVAELVRPGASVTLSCKASGYIFTDYEIHWVKQTPVHGLEWIGVIDPETGNTAFNQKFKGKATLTADISSSTAYMELSSLTSEDSAVYYCMGYSDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCEFTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.: 7GATGTTTTGATGACCCAAACTCCACGCTCCCTGTCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGTAGATCGAGTCAGAGCCTTTTACATAGTAATGGAAACACCTATTTAGAATGGTATTTGCAGAAACCAGGCCAGCCTCCAAAGGTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCGGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGCTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGTSEQ ID NO.: 8DVLMTQTPRSLSVSLGDQASISCRSSQSLLHSNGNTYLEWYLQKPGQPPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISGVEAEDLGVYYCFQGSHVPLTFGAGTKLELKAVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.: 9GAGATCCAGCTGCAGCAGTCTGGACCTGAGTTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACCTTCACTGACAACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTTACTATGGTACTACTACCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCCGCACAGCCTACATGGAGCTCCGCGGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGATGACTGGTTTGATTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCTCAACGAAGGGCCCATCTGTCTTTCCCCTGGCCCCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGAATTCACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAA SEQ ID NO.: 10EIQLQQSGPELVKPGASVKISCKASGYTFTDNYMNWVKQSHGKSLEWIGDINPYYGTTTYNQKFKGKATLTVDKSSRTAYMELRGLTSEDSAVYYCARDDWFDYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCEFTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.: 11GACATCGTTATGTCTCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTCATAACTTTTTAAACTGGTTCCAGCAGAAACCAGGAAAATCTCCAAAGACCCTGATCTTTCGTGCAAACAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTTTGAAGATTTGGGAATTTATTCTTGTCTACAGTATGATGAGATTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAGAGCTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT SEQ ID NO.: 12DIVMSQSPSSMYASLGERVTITCKASQDIHNFLNWFQQKPGKSPKTLIFRANRLVDGVPSRFSGSGSGQDYSLTISSLEFEDLGIYSCLQYDEIPLTFGAGTKLELRAVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.: 13GAGGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTTCTCCATCACCAGTGGTTATGGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAACTACGATGGTCACAATGACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCAAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGCAGTTACGACGGCTTATTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCTCAACGAAGGGCCCATCTGTCTTTCCCCTGGCCCCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGAATTCACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAA SEQ ID NO.: 14EVQLQESGPDLVKPSQSLSLTCTVTGFSITSGYGWHWIRQFPGNKLEWMGYINYDGHNDYNPSLKSRISITQDTSKNQFFLQLNSVTTEDTATYYCASSYDGLFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCEFTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.: 15GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAATAGGACAGAAGGTCACTATGAACTGCAAGTCCAGTCAGAGCCTTTTAAATAGTAACTTTCAAAAGAACTTTTTGGCCTGGTACCAGCAGAAACCAGGCCAGTCTCCTAAACTTCTGATATACTTTGCATCCACTCGGGAATCTAGTATCCCTGATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGATTACTTCTGTCAGCAACATTATAGCACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA SEQ ID NO.: 16DIVMTQSPSSLAVSIGQKVTMNCKSSQSLLNSNFQKNFLAWYQQKPGQSPKLLIYFASTRESSIPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELK SEQ ID NO.: 17GAGGTTCAGCTGCAGCAGTCTGTAGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACATATTTACTGACTATGAGATACACTGGGTGAAGCAGACTCCTGTGCATGGCCTGGAATGGATTGGGGTTATTGATCCTGAAACTGGTAATACTGCCTTCAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACATATCCTCCAGCACAGCCTACATGGAACTCAGCAGTTTGACATCTGAGGACTCTGCCGTCTATTACTGTATGGGTTATTCTGATTATTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO.: 18EVQLQQSVAELVRPGASVTLSCKASGYIFTDYEIHWVKQTPVHGLEWIGVIDPETGNTAFNQKFKGKATLTADISSSTAYMELSSLTSEDSAVYYCMGYSDYWGQGTTLTVSS SEQ ID NO.: 19GATGTTTTGATGACCCAAACTCCACGCTCCCTGTCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGTAGATCGAGTCAGAGCCTTTTACATAGTAATGGAAACACCTATTTAGAATGGTATTTGCAGAAACCAGGCCAGCCTCCAAAGGTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCGGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA SEQ ID NO.: 20DVLMTQTPRSLSVSLGDQASISCRSSQSLLHSNGNTYLEWYLQKPGQPPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISGVEAEDLGVYYCFQGSHVPLTFGAGTKLELK SEQ ID NO.: 21GAGATCCAGCTGCAGCAGTCTGGACCTGAGTTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACCTTCACTGACAACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTTACTATGGTACTACTACCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCCGCACAGCCTACATGGAGCTCCGCGGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGATGACTGGTTTGATTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA SEQ ID NO.: 22EIQLQQSGPELVKPGASVKISCKASGYTFTDNYMNWVKQSHGKSLEWIGDINPYYGTTTYNQKFKGKATLTVDKSSRTAYMELRGLTSEDSAVYYCARDDWFDYWGQGTLVTVSA SEQ ID NO.: 23GACATCGTTATGTCTCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTCATAACTTTTTAAACTGGTTCCAGCAGAAACCAGGAAAATCTCCAAAGACCCTGATCTTTCGTGCAAACAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTTTGAAGATTTGGGAATTTATTCTTGTCTACAGTATGATGAGATTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAGASEQ ID NO.: 24DIVMSQSPSSMYASLGERVTITCKASQDIHNFLNWFQQKPGKSPKTLIFRANRLVDGVPSRFSGSGSGQDYSLTISSLEFEDLGIYSCLQYDEIPLTFGAGTKLELR SEQ ID NO.: 25GAGGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTTCTCCATCACCAGTGGTTATGGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAACTACGATGGTCACAATGACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCAAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGCAGTTACGACGGCTTATTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA SEQ ID NO.: 26EVQLQESGPDLVKPSQSLSLTCTVTGFSITSGYGWHWIRQFPGNKLEWMGYINYDGHNDYNPSLKSRISITQDTSKNQFFLQLNSVTTEDTATYYCASSYDGLFAYWGQGTLVTVSA SEQ ID NO.: 27KSSQSLLNSNFQKNFLA SEQ ID NO.: 28 FASTRES SEQ ID NO.: 29 QQHYSTPLTSEQ ID NO.: 30 GYIFTDYEIH SEQ ID NO.: 31 VIDPETGNTA SEQ ID NO.: 32MGYSDY SEQ ID NO.: 33 RSSQSLLHSNGNTYLE SEQ ID NO.: 34 KVSNRFSSEQ ID NO.: 35 FQGSHVPLT SEQ ID NO.: 36 GYTFTDNYMN SEQ ID NO.: 37DINPYYGTTT SEQ ID NO.: 38 ARDDWFDY SEQ ID NO.: 39 KASQDIHNFLNSEQ ID NO.: 40 RANRLVD SEQ ID NO.: 41 LQYDEIPLT SEQ ID NO.: 42GFSITSGYGWH SEQ ID NO.: 43 YINYDGHND SEQ ID NO.: 44 ASSYDGLFAYSEQ ID NO.: 45 - 3A4 heavy chain variable region nucleotide sequenceCAGATCCAGTTGGTGCAATCTGGACCTGAGATGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACGACTACATGAGCTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTTACAACGGTGATACTAACTACAACCAGAAGTTCAAGGGCAAGGCCATATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCGGAAGACTCAGCAGTCTATTACTGTGCAAGAGACCCGGGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCASEQ ID NO.: 46 - 3A4 heavy chain variable region polypeptide sequenceQIQLVQSGPEMVKPGASVKMSCKASGYTFTDDYMSWVKQSHGKSLEWIGDINPYNGDTNYNQKFKGKAILTVDKSSSTAYMQLNSLTSEDSAVYYCARDPGAMDYWGQGTSVTVSSSEQ ID NO.: 47 - 3A4 light chain variable region nucleotide sequenceGATGTTGTGATGACCCAAACTCCACTCTCCCTGGCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTCTACATAGTAATGGAAACACCTATTTAGAATGGTACCTTCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCCACACAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAGGCTGGAGCTGAAA SEQ ID NO.: 48 - 3A4 light chain variable region polypeptidesequenceDVVMTQTPLSLAVSLGDQASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIHTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTRLELKSEQ ID NO.: 49 - 3A4 heavy chain CDR1 polypeptide sequence GYTFTDDYMSSEQ ID NO.: 50 - 3A4 heavy chain CDR2 polypeptide sequenceDINPYNGDTNYNQKFKGSEQ ID NO.: 51 - 3A4 heavy chain CDR3 polypeptide sequence DPGAMDYSEQ ID NO.: 52 - 3A4 light chain CDR1 polypeptide sequenceRSSQSLLHSNGNTYLESEQ ID NO.: 53 - 3A4 light chain CDR2 polypeptide sequence TVSNRFSSEQ ID NO.: 54 - 3A4 light chain CDR3 polypeptide sequence FQGSHVPLTSEQ ID NO.: 55 GTAAGCAGCGCTGTGGCTGCACCATCTGTCTTC SEQ ID NO.: 56GTAAGCGCTAGCCTAACACTCTCCCCTGTTGAAGC SEQ ID NO.: 57GCTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGSEQ ID NO.: 58AVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.: 59CTTGAGCCGGCGGATGGTCGAGGTGAGGTGTGGCAGGCTTGAGATCCAGCTGTTGGGGTGAGTACTCCCTCTCAAAAGCGGGCATTACTTCTGCGCTAAGATTGTCAGTTTCCAAAAACGAGGAGGATTTGATATTCACCTGGCCCGATCTGGCCATACACTTGAGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGGTCCAAGTTTAAACGGATCTCTAGCGAATTCATGAACTTTCTGCTGTCTTGGGTGCATTGGAGCCTTGCCTTGCTGCTCTACCTCCACCATGCCAAGTGGTCCCAGGCTTGAGACGGAGCTTACAGCGCTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGGGTACCGCGGCCGCTTCGAATGAGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGCCCCGCCGCCGGACGAACTAAACCTGACTACGGCATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCACGATGATAAGCTGTCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGACCAATTCTCATGTTTGACAGCTTATCATCGCAGATCCGGGCAACGTTGTTGCATTGCTGCAGGCGCAGAACTGGTAGGTATGGCAGATCTATACATTGAATCAATATTGGCAATTAGCCATATTAGTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCTCACTCTCTTCCGCATCGCTGTCTGCGAGGGCCAGCTGTTGGGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGTACTCTTGGATCGGAAACCCGTCGGCCTCCGAACGGTACTCCGCCACCGAGGGACCTGAGCGAGTCCGCATCGACCGGATCGGAAAACCTCTCGAGAAAGGCGTCTAACCAGTCACAGTCGCAAGGTAGGCTGAGCACCGTGGCGGGCGGCAGCGGGTGGCGGTCGGGGTTGTTTCTGGCGGAGGTGCTGCTGATGATGTAATTAAAGTAGGCGGT SEQ DI NO.: 60ATGCCAAGTGGTCCCAGGCTGACATTGTGATGACCCAGTCTCC SEQ ID NO.: 61ATGCCAAGTGGTCCCAGGCTGATGTTTTGATGACCCAAACTCC SEQ ID NO.: 62ATGCCAAGTGGTCCCAGGCTGACATCGTTATGTCTCAGTCTCC SEQ ID NO.: 63GGGAAGATGAAGACAGATGGTGCAGCCACAGC SEQ ID NO.: 64GTAAGCGCTAGCGCCTCAACGAAGGGCCCATCTGTCTTTCCCCTGGCCCC SEQ ID NO.: 65GTAAGCGAATTCACAAGATTTGGGCTCAACTTTCTTG SEQ ID NO.: 66GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCAGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT SEQ ID NO.: 67ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO.: 68CTTGAGCCGGCGGATGGTCGAGGTGAGGTGTGGCAGGCTTGAGATCCAGCTGTTGGGGTGAGTACTCCCTCTCAAAAGCGGGCATTACTTCTGCGCTAAGATTGTCAGTTTCCAAAAACGAGGAGGATTTGATATTCACCTGGCCCGATCTGGCCATACACTTGAGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGGTCCAAGTTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGCGGAGACGGAGCTTACGGGCCCATCTGTCTTTCCCCTGGCCCCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGAATTCACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAATGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGCCCCGCCGCCGGACGAACTAAACCTGACTACGGCATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCACGATGATAAGCTGTCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCTCACTCTCTTCCGCATCGCTGTCTGCGAGGGCCAGCTGTTGGGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGTACTCTTGGATCGGAAACCCGTCGGCCTCCGAACGGTACTCCGCCACCGAGGGACCTGAGCGAGTCCGCATCGACCGGATCGGAAAACCTCTCGAGAAAGGCGTCTAACCAGTCACAGTCGCAAGGTAGGCTGAGCACCGTGGCGGGCGGCAGCGGGTGGCGGTCGGGGTTGTTTCTGGCGGAGGTGCTGCTGATGATGTAATTAAAGTAGGCGGT SEQ ID NO.: 69GGGTTCCAGGTTCCACTGGCGAGGTTCAGCTGCAGCAGTCTGT SEQ ID NO.: 70GGGTTCCAGGTTCCACTGGCGAGGTGCAGCTTCAGGAGTCAGG SEQ ID NO: 71GGGGCCAGGGGAAAGACAGATGGGCCCTTCGTTGAGGCSEQ ID NO.: 89: Exemplary embodiment of CDRL1K-S-S-Q-S-L-L-N/H-S/T-S/N/D-N/G-Q/N/K-K/L-N-Y-L-ASEQ ID NO.: 90: Exemplary embodiment of CDRL1 K-A-S-Q-D-I-H-N/T-Y/F-L-NSEQ ID NO.: 91: Exemplary embodiment of CDRL2 F-A-S-T-R-E-SSEQ ID NO.: 92: Exemplary embodiment of CDRL2 L-V-S-K-L-D-SSEQ ID NO.: 93: Exemplary embodiment of CDRL2 R-A-N-R-L-V-DSEQ ID NO.: 94: Exemplary embodiment of CDRL3 Q-Q-H-Y-S-T-P-L-TSEQ ID NO.: 95: Exemplary embodiment of CDRL3W/L-Q-Y/G-D/T-A/E/H-F-P-R-TSEQ ID NO.: 96: Exemplary embodiment of CDRH1 1G-Y-T/I-F-T-D/E-Y-E/N-M/I/V-HSEQ ID NO.: 97: Exemplary embodiment of CDRH1 G-F-T/S-I-T-S-G-Y-G-W-HSEQ ID NO.: 98: Exemplary embodiment of CDRH2V/N/G-I/L-D-P-E/A/G-T/Y-G-X-T-ASEQ ID NO.: 99: Exemplary embodiment of CDRH2 Y-I-N/S-F/Y-N/D-GSEQ ID NO.: 100: Exemplary embodiment of CDRH3 M-G-Y-S/A-D-YSEQ ID NO.: 101: Exemplary embodiment of CDRH3 A-S-S-Y-D-G-F-L-A-YSEQ ID NO.: 102: Exemplary embodiment of CDRH3 3 A-R/W-W/F-G-L-R-Q/NSEQ ID NO. 103- 3A2 light chain variable regionDAVMTQIPLTLSVTIGQPASLSCKSSQSLLHSDGKTYLNWLLQRPGQSPKRLISLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGLYYCWQGTHFPRTFAGGTNLEIKSEQ ID NO. 104 3F6 light chain variable regionSIVMTQTPLTLSVTIGQPASITCKSSQSLLYSDGKTYLNWLLQRPGQSPKRLISLVSKLDSGVPDGFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKSEQ ID NO. 105- 3E8 light chain variable regionDAVMTQIPLTLSVTIGQPASISCKSSQSLLHSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIKSEQ ID NO. 106- 3E10 light chain variable regionDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIKSEQ ID NO. 107- 3A9 light chain variable regionDIVMTQSPSSLAMSLGQKVTMSCKSSQSLLNSNNQLNYLAWYQQKPGQSPKLLVYFASTRKSGVPDRFIGSGSGTDFTLTITSVQAEDLADYFCQQHFNTPLTFGAGTKLELKSEQ ID NO. 108- 3B1 light chain variable regionDIVMTQSPSSLAISVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVFFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSIPLTFGAGTKLELKSEQ ID NO. 109- 3G5 light chain variable regionDIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVFFASTRESGVPDRFIGSGSGTDFTLTITSVQAEDLADYFCQQHYSIPLTFGSGTKLELKSEQ ID NO. 110- 3B2 light chain variable regionDIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSIPLTFGAGTKLELKSEQ ID NO. 111- 3B8 light chain variable regionDIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELKSEQ ID NO. 112- 3G8 light chain variable regionDIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELKSEQ ID NO. 113- 3F7 light chain variable regionDIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLIYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELKSEQ ID NO. 114- 3E9 light chain variable regionDIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTEFTLTITSVQAEDLADYFCQQHYSTPLTFGAGTKLELKSEQ ID NO. 115- 3C3 light chain variable regionDIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFGSTRESGVPDRFIGSGSGTDFTLTISGVQAEDLADYFCQQHYSTPLTFGAGTKLELKSEQ ID NO. 116- 3E12 light chain variable regionDIVMTQSPSSLAMSVGQKVTMNCKSSQSLLNRSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSIPLTFGAGTKLELKSEQ ID NO. 117- 4A2 light chain variable regionDIVMTQSPSSLAMSVGQKVTMNCKSSQSLLNNSNQKNYLAWYQQKPGQSPKLLLYFASTRESGVPDRFIGSGSGTYFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLDLKSEQ ID NO. 118- 3F10 light chain variable regionDIVMTQSPSSLTMSVGQKVTMSCKSSQSLLNTSNQLNYLAWYQQKPGQSPKLLVYFASTTESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELKSEQ ID NO. 119- 3F4 light chain variable regionDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNTSNQKNYLAWYQQKPGQSPKLLVYFASTRASGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELKSEQ ID NO. 120- 3B11 light chain variable regionDIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELKSEQ ID NO. 121- 3G12 light chain variable regionDIVMTQSPKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPELLIYWTSTRHTGVPDRFSGSGSGTDFTLTISSVQAEDLADYFCQQHYSIPLTFGAGTKLELRSEQ ID NO. 122- 3D1 light chain variable regionDIKMTQSPSSMYASLGERVTITCKASQDIHTYLNWFQQKPGKSPETLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELKSEQ ID NO. 123- 3C2 light chain variable regionDIQMTQSPSSMYASLGERVTLTCKASQDIHNYLNWFQQKPGKSPKTLIHRANRLVAGVPSRFSGSGSGQDYSLTISSLEYEDLGIYYCLQYDAFPLTFGAGTKLELKSEQ ID NO. 124- 3E6 light chain variable regionDIQMTQSPSSMYASLGERVTLTCKASQDIHNYLNWFQQKPGKSPKTLIHRANRLVAGVPSRFSGSGSGQDYSLTISSLEYEDLGIYYCLQYDAFPLTFGAGTKLELKSEQ ID NO. 125- 3H3 light chain variable regionDIVMSQSPSSMYASLGERVTITCKASQDIHRFLNWFQQKPGKSPKTLIFHANRLVDGVPSRFSGSGSGLDYSLTISSLEYEDMGIYFCLQYDAFPLTFGAGTKLELKSEQ ID NO. 126- 3A2 heavy chain variable regionHEIQLQQSGPELVKPGASVKMSCKTSGYTFTDYNMHWVKQKPGQGLEWIGYINPYNDVTEYNEKFKGRATLTSDKSSSTAYMDLSSLTSDDSAVYFCAWFGLRQWGQGTLVTVSTSEQ ID NO. 127- 3F6 heavy chain variable regionHEVQLQQSGPELVKPGASVKMSCKASGYIFTEYNIHWVKQKPGQGPEWIGNINPYNDVTEYNEKFKGKATLTSDKASSTAYMDLSSLTSEDSAVYYCARWGLRNWGQGTLVTVSASEQ ID NO. 128- 3E8 heavy chain variable regionHEVQLQQSVPELVKPGASVKMSCKTSGYTFTEYNMHWVKQKPGQGPEWIGNINPYNNVTEYNEKFKGKATLTSDKSSSTAYLDLSSLTSEDSAVYYCARWGLRNWGQGTLVTVSASEQ ID NO. 129- 3A9 heavy chain variable regionHQVQVQQPGAELVRPGASVTLSCKASGYIFTDYEVHWVRQRPVHGLEWIGVIDPETGDTAYNQKFKGKATLTADKSSSTAYMELSSLTAEDSAVYYCIGYADYWGQGTTLTVSSSEQ ID NO. 130- 3B1 heavy chain variable regionHQVQLQQPGAELVRPGASVTLSCKASGYTFTDYEIHWVKQTPVHGLEWIGVIDPETGGTAYNQKFKGKATLTTDKSSSTAYMELRSLTSEDSAVYYCMGYSDYWGQGTTLTVSSSEQ ID NO. 131- 3B2 heavy chain variable regionHEVQLQQSGAELVRPGASVTLSCKASGYTFTDYEIHWVKQTPVHGLEWIGVIDPETGATAYNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYCMGYSDYWGQGTTLTVSSSEQ ID NO. 132- 3F4 heavy chain variable regionHEVQLQQSGAELVRPGASVTLSCKASGYTFTDYEIHWVKQTPVHGLEWIGVIDPETGSTAYNQKFKGKATLTADKASSTAYMELSSLTSEDSAVYYCMGYSDYWGQGTTLTVSSSEQ ID NO. 133- 3E9 heavy chain variable regionHEVQLQQSGAELVRPGASATLSCKASGYTFTDYEMHWVKQTPVHGLEWIGVIDPETGSTAYNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYCMGYADYWGQGTTLTVSSSEQ ID NO. 134- 3B8 heavy chain variable regionHEVQLQQSGAELVRPGASVTLSCKASGYTFTDYEIHWVKQTPVHGLEWIGVIDPETGDTAYNQNFTGKATLTADKSSSTAYMELSSLTSEDSAVYYCMGYADYWGQGTTLTVSSSEQ ID NO. 135- 3G8 heavy chain variable regionHQVQLKQSGAELVRPGASVTLSCKASGYTFTDYEVHWVKQTPVHGLEWIGVIDPATGDTAYNQKFKGKATLTADKSSSTAYMEVSSLTSEDSAVYYCMGYSDYWGQGTTLTVSSSEQ ID NO. 136- 3F7 heavy chain variable regionHQAYLQQSGAELVRPGASVTLSCKASGYTFTDYEIHWVKQTPVHGLEWIGVIDPETGDTAYNQKFKDKATLTADKASSTAYMELSSLTSEDSAVYYCMGYSDYWGQGTTLTVSSSEQ ID NO. 137- 3E12 heavy chain variable regionHQVQLQQSEAELVKPGASVKLSCKASGYTFTDYEIHWVKQTPVHGLEWIGVIDPETGDTAYNQKFKGKATLTADKSSSTAYMELSRLTSEDSAVYYCMGHSDYWGQGTTLTVSSSEQ ID NO. 138- 3G12 heavy chain variable regionHEVQLQQSVAELVRPGASVTVSCKASGYIFTDYEIHWVKQTPAHGLEWIGVIDPETGNTAFNQKFKGKATLTADISSSTAYMELSSLTSEDSAVYYCMGYSDYWGQGTTLTVSSSEQ ID NO. 139- 3F10 heavy chain variable regionHEVQLQQSVAELVRPGAPVTLSCKASGYTFTDYEVHWVKQTPVHGLEWIGVIDPETGATAYNQKFKGKATLTADKSSSAAYMELSRLTSEDSAVYYCMSYSDYWGQGTTLTVSSSEQ ID NO. 140- 3C3 heavy chain variable regionHEVQLQQSVAEVVRPGASVTLSCKASGYTFTDYEIHWVKQTPVHGLEWIGVIDPETGVTAYNQRFRDKATLTTDKSSSTAYMELSSLTSEDSAVYFCMGYSDYWGQGTTLTVSSSEQ ID NO. 141- 3G5 heavy chain variable regionHQVQLQQPGAELVRPGASVTLSCKASGYTFTDYEIHWVKQTPVHGLEWIGVLDPGTGRTAYNQKFKDKATLSADKSSSTAYMELSSLTSEDSAVYYCMSYSDYWGPGTTLTVSSSEQ ID NO. 142- 3B11 heavy chain variable regionHEVQLQQSVAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVRGLEWIGVIDPATGDTAYNQKFKGKATLTADKSSSAAFMELSSLTSEDSAVYYCMGYSDYWGQGTTLTVSSSEQ ID NO. 143- 3E6 heavy chain variable regionHQVQLQQSGAELVRPGASVTLSCKASGYTFSDYEMHWVKQTPVHGLEWIGGIDPETGDTVYNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYCISYAMDYWGQGTSVTVSSSEQ ID NO. 144- 4A2 heavy chain variable regionHQVKLQQSGTELVRPGASVTLSCKASGYKFTDYEMHWVKQTPVHGLEWIGGIDPETGGTAYNQKFKGKAILTADKSSTTAYMELRSLTSEDSAVYYCISYAMDYWGQGTSVTVSSSEQ ID NO. 145- 3E10 heavy chain variable regionHEVQLQQSGPELVKPGASVKISCKASGDTFTDYYMNWVKQSHGKSLEWIGDINPNYGGITYNQKFKGKATLTVDTSSSTAYMELRGLTSEDSAVYYCQAYYRNSDYWGQGTTLTVSSSEQ ID NO. 146- 3D1 heavy chain variable regionHEVQLQESGPDLVKPSQSLSLTCTVTGFSITSGYGWHWIRQFPGDKLEWMGYISFNGDYNYNPSLKSRISITRDTSKNQFFLQLSSVTTEDTATYYCASSYDGLFAYWGQGTLVTVSASEQ ID NO. 147- 3C2 heavy chain variable regionHDVQLQESGPDLVKPSQSLSLTCTVTGFSITSGYGWHWIRQFPGNKLEWMGYISFNGDSNYNPSLKSRISITRDTSKNQFFLQLNSVTSEDTATYYCASSYDGLFAYWGQGPLVTVSA A SEQ ID NO.: 148KSSQSLLHSDGKTYLN SEQ ID NO.: 149 LVSKLDS SEQ ID NO.: 150 WQGTHFPRTSEQ ID NO.: 151 GYTFTD YNMH SEQ ID NO.: 152 YINPYNDVTE SEQ ID NO.: 153AWFGL RQ SEQ ID NO.: 154 RSSKSLLHSNGN TYLY SEQ ID NO.: 155 RMSNLASSEQ ID NO.: 156 MQHLEYPYT SEQ ID NO.: 157 GDTFTD YYMN SEQ ID NO.: 158DINPNYGGIT SEQ ID NO.: 159 QAYYRNS DY SEQ ID NO.: 160 KASQDVGTAVASEQ ID NO.: 161 WTSTRHT SEQ ID NO.: 162 QQHYSIPLT SEQ ID NO.: 163GYIFTDYEIH SEQ ID NO.: 164 VIDPETGNTA SEQ ID NO.: 165 MGYSDYSEQ ID NO.: 166MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCKSSQSLLNSNFQKNFLAWYQQKPGQPPKLLIYFASTRESSVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.: 167MDWTWRILFLVAAATGTHAEVQLVQSGAEVKKPGASVKVSCKASGYIFTDYEIHWVRQAPGQGLEWMGVIDPETGNTAFNQKFKGRVTITADTSTSTAYMELSSLTSEDTAVYYCMGYSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID N: 168DIVMTQSPDSLAVSLGERATINCKSSQSLLNSNFQKNFLAWYQQKPGQPPKLLIYFASTRESSVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTKLEIK SEQ ID NO.: 169EVQLVQSGAEVKKPGASVKVSCKASGYIFTDYEIHWVRQAPGQGLEWMGVIDPETGNTAFNQKFKGRVTITADTSTSTAYMELSSLTSEDTAVYYCMGYSDYWGQGTLVTVSS SEQ ID NO.: 170MVLQTQVFISLLLWISGAYGDIVMTQSPSSLSASVGDRVTITCKASQDIHNFLNWFQQKPGKAPKTLIFRANRLVDGVPSRFSGSGSGTDYTLTISSLQPEDFATYSCLQYDEIPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.: 171MDWTWRILFLVAAATGTHAEVQLQESGPGLVKPSQTLSLTCTVSGFSITSGYGWHWIRQHPGKGLEWIGYINYDGHNDYNPSLKSRVTISQDTSKNQFSLKLSSVTAADTAVYYCASSYDGLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No.: 172DIVMTQSPSSLSASVGDRVTITCKASQDIHNFLNWFQQKPGKAPKTLIFRANRLVDGVPSRFSGSGSGTDYTLTISSLQPEDFATYSCLQYDEIPLTFGQGTKLEIK SEQ ID NO.: 173EVQLQESGPGLVKPSQTLSLTCTVSGFSITSGYGWHWIRQHPGKGLEWIGYINYDGHNDYNPSLKSRVTISQDTSKNQFSLKLSSVTAADTAVYYCASSYDGLFAYWGQGTLVTVSSEQ ID NO.: 186 (3A4 variant light chain variable region consensus 1)DXVMTQTPLSLXVXXGXXASISCRSSQSLLHSNGNTYLEWYLQKPGQSPXLLIHTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDXGVYYCFQGSHVPLTFGXGTXLEXKwherein at least one of the amino acids identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:48.The amino acid substitution may be, for example conservative.

(3A4 variant light chain variable region consensus 2) SEQ ID NO.: 187DX_(a1)VMTQTPLSLX_(a2)VX_(a3)X_(a4)GX_(a5)X_(a6)ASISCRSSQSLLHSNGNTYLEWYLQKPGQSPX_(a7)LLIHTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDX_(a8)GVYYCFQGSHVPLTFGX_(a9)GTX_(a10)LEX_(a11)KWherein X_(a1) may be a hydrophobic amino acid;

Wherein X_(a2) may be A or P;

Wherein X_(a3) may be neutral hydrophilic amino acid;

Wherein X_(a4) may be L or P;

Wherein X_(a5) may be an acidic amino acid;

Wherein X_(a6) may be Q or P;

Wherein X_(a7) may be a basic amino acid;Wherein X_(a8) may be a hydrophobic amino acid;

Wherein X_(a9) may be A or Q;

Wherein X_(a10) may be a basic amino acid; orWherein X_(a11) may be a hydrophobic amino acid,wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:48.

(3A4 variant light chain variable region consensus 3) SEQ ID NO.: 188DX_(A1)VMTQTPLSLX_(A2)VX_(A3)X_(A4)GX_(A5)X_(A6)ASISCRSSQSLLHSNGNTYLEWYLQKPGQSPX_(A7)LLIHTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDX_(A8)GVYYCFQGSHVPLIFGX_(A9)GTX_(A10)LEX_(A11)K

Wherein X_(A1) may be V or I Wherein X_(A2) may be A or P Wherein X_(A3)may be S or T Wherein X_(A4) may be L or P Wherein X_(A5) may be D or EWherein X_(A6) may be Q or P Wherein X_(A7) may be K or Q Wherein X_(A8)may be L or V Wherein X_(A9) may be A or Q Wherein X_(A10) may be R or Kor Wherein X_(A11) may be L or I,

wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:48.

(3A4 variant 1 light chain variable region: Lvh1) SEQ ID NO.: 189DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP LTFGQGTKLEIK(3A4 variant 2 light chain variable region: Lvh2) SEQ ID NO.: 190DVVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP LTFGQGTKLEIK(3A4 variant heavy chain variable region consensus 1) SEQ ID NO.: 191QXQLVQSGXEXXKPGASVKXSCKASGYTFTDDYMSWVXQXXGXXLEWXGDINPYNGDTNYNQKFKGXXXXTXDXSXSTAYMXLXSLXSEDXAVYYCARDP GAMDYWGQGTXVTVSSwherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:46.The amino acid substitution may be, for example conservative.

(3A4 variant heavy chain variable region consensus 2) SEQ ID NO.: 192QX_(b1)QLVQSGX_(b2)EX_(b3)X_(b4)KPGASVKX_(b5)SCKASGYTFTDDYMSWVX_(b6)QX_(b7)X_(b8)GX_(b9)X_(b10)LEWX_(b11)GDINPYNGDTNYNCKPKGX_(b12)X_(b13)X_(b14)X_(b15)TX_(b16)DX_(b17)SX_(b18)STAYMX_(b19)LX_(b20)SLX_(b21)SEDX_(b22)AVYYCARDPGAMDYWGQGTX_(b23)VTVSSWherein X_(b1) may be a hydrophobic amino acid;

Wherein X_(b2) may be P or A;

Wherein X_(b3) may be a hydrophobic amino acid;

Wherein X_(b4) may be V or K;

Wherein X_(b5) may be a hydrophobic amino acid;Wherein X_(b6) may be a basic amino acid;

Wherein X_(b7) may be S or A; Wherein X_(b8) may be H or P;

Wherein X_(b9) may be a basic amino acid;

Wherein X_(b10) may be S or G;

Wherein X_(b11) may be a hydrophobic amino acid;Wherein X_(b12) may be a basic amino acid;Wherein X_(b13) may be a hydrophobic amino acid;

Wherein X_(b14) may be I or T;

Wherein X_(b15) may be a hydrophobic amino acid;Wherein X_(b16) may be a hydrophobic amino acid;

Wherein X_(b17) may be K or T;

Wherein X_(b18) may be a neutral hydrophilic amino acid;

Wherein X_(b19) may be Q or E; Wherein X_(b20) may be N or S; WhereinX_(b21) may be T or R;

Wherein X_(b22) may be a neutral hydrophilic amino acid; or

Wherein X_(b23) may be S or L,

wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:46.

(3A4 variant heavy chain variable region consensus 3) SEQ ID NO.: 193QX_(B1)QLVQSGX_(B2)EX_(B3)X_(B4)KPGASVKX_(B5)SCKASGYTFTDDYMSWVX_(B6)QX_(B7)X_(B8)GX_(B9)X_(B10)LEWX_(B11)GDINPYNGDTNYNQKFKGX_(B12)X_(B13)X_(B14)X_(B15)TX_(B16)DX_(B17)SX_(B18)STAYMX_(B19)LX_(B20)SLX_(B21)SEDX_(B22)AVYYCARDPGAMDYWGQGTX_(B23)VTVSS

Wherein X_(B1) may be I or V; Wherein X_(B2) may be P or A; WhereinX_(B3) may be M or V; Wherein X_(B4) may be V or K; Wherein X_(B5) maybe M or V; Wherein X_(B6) may be K or R; Wherein X_(B7) may be S or A;Wherein X_(B8) may be H or P; Wherein X_(B9) may be K or Q; WhereinX_(B10) may be S or G; Wherein X_(B11) may be I or M; Wherein X_(B12)may be K or R; Wherein X_(B13) may be A or V; Wherein X_(B14) may be Ior T; Wherein X_(B15) may be L or I; Wherein X_(B16) may be V or A;Wherein X_(B17) may be K or T; Wherein X_(B18) may be S or T; WhereinX_(B19) may be Q or E; Wherein X_(B20) may be N or S; Wherein X_(B21)may be T or R; Wherein X_(B22) may be S or T; or Wherein X_(B23) may beS or L,

wherein at least one of the amino acid identified by X is an amino acidsubstitution (conservative or non-conservative) in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:46.

(3A4 variant 1 heavy chain variable region: Hvh1) SEQ ID NO.: 194QVQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWMGDINPYNGDTNYNQKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSS(3A4 variant 2 heavy chain variable region: Hvh2) SEQ ID NO.: 195QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWMGDINPYNGDTNYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSS(3A4 variant 3 heavy chain variable region: Hvh3) SEQ ID NO.: 196QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWIGDINPYNGDTNYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSS(3A4 variant 4 heavy chain variable region: Hvh4) SEQ ID NO.: 197QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGDTNYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSS3A4 murine light (kappa) chain SEQ ID NO: 198DVVMTQTPLSLAVSLGDQASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIHTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTRLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC3A4 humanized light (kappa) chain variant 1; Lh1 SEQ ID NO: 199DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC3A4 humanized light (kappa) chain variant 2; Lh2 SEQ ID NO: 200DVVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 3A4 murine heavy (Igg1) chain SEQ ID NO: 201QIQLVQSGPEMVKPGASVKMSCKASGYTFTDDYMSWVKQSHGKSLEWIGDINPYNGDTNYNQKFKGKAILTVDKSSSTAYMQLNSLTSEDSAVYYCARDPGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K3A4 humanized heavy (Igg1) chain variant 1; Hh1 SEQ ID NO: 202QVQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWMGDINPYNGDTNYNQKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K3A4 humanized heavy (Igg1) chain variant 2; Hh2 SEQ ID NO: 203QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWMGDINPYNGDTNYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK3A4 humanized heavy (Igg1) chain variant 3; Hh3 SEQ ID NO: 204QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWIGDINPYNGDTNYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK3A4 humanized heavy (Igg1) chain variant 4: Hh4 SEQ ID NO: 205QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGDTNYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 206 ATACCCAAGCTTGCCACCATGGAGACAGACACAC SEQ ID NO: 207ATACCCAAGCTTCATTTCCCGGGAGACAGGGAG SEQ ID NO: 208ATACCCAAGCTTGGGCCACCATGAACTTTCTGCTGTCTTGG SEQ ID NO: 209ATACCCAAGCTTCTAACACTCTCCCCTGTTGAAG pK-CR5 SEQ ID NO: 210CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCACATCGGCGCGCCAAATGATTTGCCCTCCCATATGTCCTTCCGAGTGAGAGACACAAAAAATTCCAACACACTATTGCAATGAAAATAAATTTCCTTTATTAGCCAGAGGTCGAGATTTAAATAAGCTTGCTAGCAGATCTTTGGACCTGGGAGTGGACACCTGTGGAGAGAAAGGCAAAGTGGATGTCATTGTCACTCAAGTGTATGGCCAGATCGGGCCAGGTGAATATCAAATCCTCCTCGTTTTTGGAAACTGACAATCTTAGCGCAGAAGTAATGCCCGCTTTTGAGAGGGAGTACTCACCCCAACAGCTGGATCTCAAGCCTGCCACACCTCACCTCGACCATCCGCCGTCTCAAGACCGCCTACTTTAATTACATCATCAGCAGCACCTCCGCCAGAAACAACCCCGACCGCCACCCGCTGCCGCCCGCCACGGTGCTCAGCCTACCTTGCGACTGTGACTGGTTAGACGCCTTTCTCGAGAGGTTTTCCGATCCGGTCGATGCGGACTCGCTCAGGTCCCTCGGTGGCGGAGTACCGTTCGGAGGCCGACGGGTTTCCGATCCAAGAGTACTGGAAAGACCGCGAAGAGTTTGTCCTCAACCGCGAGCCCAACAGCTGGCCCTCGCAGACAGCGATGCGGAAGAGAGTGACCGCGGAGGCTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCGTGGATGGCGTCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGGCCTCCCACCGTACACGCCTACCTCGACCCGGGTACCAATCTTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTAAGGTTGTCGAGTGAAGACGAAAGGGTTCATTAAGGCGCGCCGTCGACCTCGAGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC pMPG-CR5 SEQ ID NO: 211GTCGACGATACCGTGCACTTAATTAAGCGCGCTCGACCAAATGATTTGCCCTCCCATATGTCCTTCCGAGTGAGAGACACAAAAAATTCCAACACACTATTGCAATGAAAATAAATTTCCTTTATTAGCCAGAGGTCGAGGTCGGGGGATCCGTTTAAACTTGGACCTGGGAGTGGACACCTGTGGAGAGAAAGGCAAAGTGGATGTCATTGTCACTCAAGTGTATGGCCAGATCGGGCCAGGTGAATATCAAATCCTCCTCGTTTTTGGAAACTGACAATCTTAGCGCAGAAGTAATGCCCGCTTTTGAGAGGGAGTACTCACCCCAACAGCTGGATCTCAAGCCTGCCACACCTCACCTCGACCATCCGCCGTCTCAAGACCGCCTACTTTAATTACATCATCAGCAGCACCTCCGCCAGAAACAACCCCGACCGCCACCCGCTGCCGCCCGCCACGGTGCTCAGCCTACCTTGCGACTGTGACTGGTTAGACGCCTTTCTCGAGAGGTTTTCCGATCCGGTCGATGCGGACTCGCTCAGGTCCCTCGGTGGCGGAGTACCGTTCGGAGGCCGACGGGTTTCCGATCCAAGAGTACTGGAAAGACCGCGAAGAGTTTGTCCTCAACCGCGAGCCCAACAGCTGGCCCTCGCAGACAGCGATGCGGAAGAGAGTGACCGCGGAGGCTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCGTGGATGGCGTCTCCAGGCGATCTGACGGITCACTAAACGAGCTCTGCTTATATAGGCCTCCCACCGTACACGCCTACCTCGACCCGGGTACCAATCTTATAATACAAACAGACCAGATIGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTAAGGTTGTCGAGTGAAGACGAAAGGGTTAATTAAGGCGCGCCGTCGACTAGCTTGGCACGCCAGAAATCCGCGCGGTGGTTTTTGGGGGTCGGGGGTGTTTGGCAGCCACAGACGCCCGGTGTTCGTGTCGCGCCAGTACATGCGGTCCATGCCCAGGCCATCCAAAAACCATGGGTCTGTCTGCTCAGTCCAGTCGTGGACCAGACCCCACGCAACGCCCAAAATAATAACCCCCACGAACCATAAACCATTCCCCATGGGGGACCCCGTCCCTAACCCACGGGGCCAGTGGCTATGGCAGGGCCTGCCGCCCCGACGTTGGCTGCGAGCCCTGGGCCTTCACCCGAACTTGGGGGGTGGGGTGGGGAAAAGGAAGAAACGCGGGCGTATTGGCCCCAATGGGGTCTCGGTGGGGTATCGACAGAGTGCCAGCCCTGGGACCGAACCCCGCGTTTATGAACAAACGACCCAACACCCGTGCGTTTTATTCTGTCTTTTTATTGCCGTCATAGCGCGGGTTCCTTCCGGTATTGTCTCCTTCCGTGTTTCAGTTAGCCTCCCCCATCTCCCCTATTCCTTTGCCCTCGGACGAGTGCTGGGGCGTCGGTTTCCACTATCGGCGAGTACTTCTACACAGCCATCGGTCCAGACGGCCGCGCTTCTGCGGGCGATTTGTGTACGCCCGACAGTCCCGGCTCCGGATCGGACGATTGCGTCGCATCGACCCTGCGCCCAAGCTGCATCATCGAAATTGCCGTCAACCAAGCTCTGATAGAGTTGGTCAAGACCAATGCGGAGCATATACGCCCGGAGCCGCGGCGATCCTGCAAGCTCCGGATGCCTCCGCTCGAAGTAGCGCGTCTGCTGCTCCATACAAGCCAACCACGGCCTCCAGAAGAAGATGTTGGCGACCTCGTATTGGGAATCCCCGAACATCGCCTCGCTCCAGTCAATGACCGCTGTTATGCGGCCATTGTCCGTCAGGACATTGTTGGAGCCGAAATCCGCGTGCACGAGGTGCCGGACTTCGGGGCAGTCCTCGGCCCAAAGCATCAGCTCATCGAGAGCCTGCGCGACGGACGCACTGACGGTGTCGTCCATCACAGTTTGCCAGTGATACACATGGGGATCAGCAATCGCGCATATGAAATCACGCCATGTAGTGTATTGACCGATTCCTTGCGGTCCGAATGGGCCGAACCCGCTCGTCTGGCTAAGATCGGCCGCAGCGATCGCATCCATGGCCTCCGCGACCGGCTGCAGAACAGCGGGCAGTTCGGTTTCAGGCAGGTCTTGCAACGTGACACCCTGTGCACGGCGGGAGATGCAATAGGTCAGGCTCTCGCTGAATTCCCCAATGTCAAGCACTTCCGGAATCGGGAGCGCGGCCGATGCAAAGTGCCGATAAACATAACGATCTTTGTAGAAACCATCGGCGCAGCTATTTACCCGCAGGACATATCCACGCCCTCCTACATCGAAGCTGAAAGCACGAGATTCTTCGCCCTCCGAGAGCTGCATCAGGTCGGAGACGCTGTCGAACTTTTCGATCAGAAACTTCTCGACAGACGTCGCGGTGAGTTCAGGCTTTTTCATATCTCATTGCCCGGGATCTGCGGCACGCTGTTGACGCTGTTAAGCGGGTCGCTGCAGGGTCGCTCGGTGTTCGAGGCCACACGCGTCACCTTAATATGCGAAGTGGACCTGGGACCGCGCCGCCCCGACTGCATCTGCGTGTTCGAATTCGCCAATGACAAGACGCTGGGCGGGGTTTGTGTCATCATAGAACTAAAGACATGCAAATATATTTCTTCCGGGGACACCGCCAGCAAACGCGAGCAACGGGCCACGGGGATGAAGCAGGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCACTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTCATGTTTGACAGCTTATCTCTAGCAGATCCGGAATTCCCCTCCCCAATTTAAATGAGGACCTAACCTGTGGAAATCTACTGATGTGGGAGGCTGTAACTGTACAAACAGAGGTTATTGGAATAACTAGCATGCTTAACCTTCATGCAGGGTCACAAAAAGTGCATGACGATGGTGGAGGAAAACCTATTCAAGGCAGTAATTTCCACTTCTTTGCTGTTGGTGGAGACCCCTTGGAAATGCAGGGAGTGCTAATGAATTACAGGACAAAGTACCCAGATGGTACTATAACCCCTAAAAACCCAACAGCCCAGTCCCAGGTAATGAATACTGACCATAAGGCCTATTTGGACAAAAACAATGCTTATCCAGTTGAGTGCTGGGTTCCTGATCCTAGTAGAAATGAAAATACTAGGTATTTTGGGACTTTCACAGGAGGGGAAAATGTTCCCCCAGTACTTCATGTGACCAACACAGCTACCACAGTGTTGCTAGATGAACAGGGTGTGGGGCCTCTTTGTAAAGCTGATAGCCTGTATGTTTCAGCTGCTGATATTTGTGGCCTGTTTACTAACAGCTCTGGAACACAACAGTGGAGAGGCCTTGCAAGATATTTTAAGATCCGCCTGAGAAAAAGATCTGTAAAGAATCCTTACCTAATTTCCTTTTTGCTAAGTGACCTTATAAACAGGAGAACCCAGAGAGTGGATGGGCAGCCTATGTATGGTATGGAATCCCAGGTAGAAGAGGTTAGGGTGTTTGATGGCACAGAAAGACTTCCAGGGGACCCAGATATGATAAGATATATTGACAAACAGGGACAATTGCAAACCAAAATGCTTTAAACAGGTGCTTTTATTGTACATATACATTTAATAAATGCTGCTTTTGTATAAGCCACTTTTAAGCTTGTGTTATTTTGGGGGTGGTGTTTTAGGCCTTTTAAAACACTGAAAGCCTTTACACAAATGCAACTCTTGACTATGGGGGTCTGACCTTTGGGAATGTTCAGCAGGGGCTGAAGTATCTGAGACTTGGGAAGAGCATTGTGATTGGGATTCAGTGCTTGATCCATGTCCAGAGTCTTCAGTTTCTGAATCCTCTTCTCTTGTAATATCAAGAATACATTTCCCCATGCATATATTATATTTCATCCTTGAAAAAGTATACATACTTATCTCAGAATCCAGCCTTTCCTTCCATTCAACAATTCTAGAAGTTAAAACTGGGGTAGATGCTATTACAGAGGTAGAATGCTTCCTAAACCCAGAAATGGGGGATCTGC3A4 humanized heavy chain CDR2 polypeptide sequence SEQ ID NO.: 212DINPYNGDTN OGS18500 SEQ ID NO.: 213ATGCCAAGTGGTCCCAGGCTGATGTTGTGATGACCCAAACTCC OGS2084 SEQ ID NO:. 214GGGAAGATGAAGACAGATGGTGCAGCCACAGTCCG OGS1879 SEQ ID NO.: 215GGGTTCCAGGTTCCACTGGCCAGATCCAGTTGGTGCAATCTGG OGS1810 EQ ID NO.: 216GGGGCCAGGGGAAAGACAGATGGGCCCTTCGTTGAGGC

REFERENCES

-   Santana-Davila R. and Perez E. A. (2010) “Treatment options for    patients with triple-negative breast cancer” J Hematol Oncol. 27:42.-   de Ruijter T. C., Veeck J., et al. (2011) “Characteristics of    triple-negative breast cancer.” J Cancer Res Clin Oncol. 137:183.-   Ismail-Khan R. and Bui M. M. (2010) “A review of Triple-negative    breast cancer” Cancer Control 17:173.-   Carey L. A., Perou C. M. et al. (2006) “Race, breast cancer    subtypes, and survival in the Carolina Breast Cancer Study.” JAMA    295:2492.-   Krieg M., Seynaeve C. et al. (2009) “Sensitivity to first-line    chemotherapy for metastatic breast cancer in BRCA1 and BRCA2    mutation carriers.” J Clin Oncol 27:3764.-   Rouzier R., Perou C. M. et al. (2005) “Breast cancer molecular    subtypes respond differently to preoperative chemotherapy” Clin    Cancer Res 11:5678.-   Fong P. C., Boss D. S. et al. (2009) “Inhibition of poly(ADP-ribose)    polymerase in tumors from BRCA mutation carriers.” N Engl J Med    361:123.-   Dent R., Trudeau M et al. (2007) “Triple-Negative Breast Cancer:    Clinical Feature and Patterns of Recurrence” Clin. Cancer Res. 13:    4429.-   Bernstein L and J. V. Lacey Jr. (2011) “Receptors, Associations, and    Risk Factor Differences by Breast Cancer Subtypes: Positive or    Negative?” J Natl Cancer Inst 103(6): 451-453 (Advanced publication    Feb. 23, 2011).-   Nofech-Mozes S. et al., (2009) “Patterns of recurrence in the basal    and non-basal subtypes of triple-negative breast cancers” Cancer    Res. Treat. 118: 131-137.

1. A method of treating breast cancer, the method comprisingadministering an antibody or an antigen binding fragment thereof,capable of specific binding to Kidney associated antigen 1 (KAAG1) to anindividual having a breast cancer that has low expression of theestrogen receptor (ER), of the progesterone receptor (PgR) and/or ofhuman epidermal growth factor receptor 2 (Her2).
 2. (canceled)
 3. Themethod of claim 1, wherein the individual has a breast cancer that ischaracterized as being negative for estrogen receptor (ER) expression,progesterone receptor (PgR) expression and/or for Her2 overexpression.4. A method of treating triple negative breast cancer, the methodcomprising administering an antibody or an antigen binding fragmentthereof capable of specific binding to Kidney associated antigen 1(KAAG1) to an individual in need.
 5. (canceled)
 6. The method of claim4, wherein the antibody or antigen binding fragment thereof isconjugated with a therapeutic moiety.
 7. (canceled)
 8. The method ofclaim 4, wherein the antibody or antigen binding fragment thereof bindsan epitope comprised between amino acids 30 to 84 of KAAG1.
 9. Themethod of claim 4, wherein the antibody or antigen binding fragmentthereof is a monoclonal antibody, a chimeric antibody, a human antibodyor a humanized antibody or an antigen binding fragment thereof.
 10. Themethod of claim 4, wherein the antibody or antigen binding fragmentthereof is administered in combination with an anti-cancer agent. 11.The method of claim 4, wherein the antibody or antigen binding fragmentthereof comprises: a. a CDRH1 as set forth in SEQ ID NO.:49, a CDRH2 asset forth in SEQ ID NO.:50 or in SEQ ID NO.:212, a CDRH3 as set forth inSEQ ID NO.:51, a CDRL1 as set forth in SEQ ID NO.: 52, a CDRL2 as setforth in SEQ ID NO.:53 and a CDRL3 as set forth in SEQ ID NO.: 54; b. alight chain variable region as set forth in SEQ ID NO.:48 and a heavychain variable region as set forth in SEQ ID NO.:46; c. a light chainvariable region as set forth in SEQ ID NO.:186 wherein at least one ofthe amino acid identified by X is an amino acid substitution incomparison with a corresponding amino acid in the polypeptide set forthin SEQ ID NO.:48 and a heavy chain variable region as set forth in SEQID NO.:191 wherein at least one of the amino acid identified by X is anamino acid substitution in comparison with a corresponding amino acid inthe polypeptide set forth in SEQ ID NO.:46; d. a light chain variableregion as set forth in SEQ ID NO.:187 and a heavy chain variable regionas set forth in SEQ ID NO.:192; e. a light chain variable region as setforth in SEQ ID NO.:188 and a heavy chain variable region as set forthin SEQ ID NO.:193; f. a light chain variable region as set forth in SEQID NO.: 189 or SEQ ID NO.:190 and a heavy chain variable region as setforth in SEQ ID NO.:194, SEQ ID NO.:195, SEQ ID NO.:196 or SEQ IDNO.:197; g. a light chain variable region as set forth in SEQ ID NO.:189and a heavy chain variable region as set forth in SEQ ID NO.:194; h. alight chain variable region as set forth in SEQ ID NO.:189 and a heavychain variable region as set forth in SEQ ID NO.:195; i. a light chainvariable region as set forth in SEQ ID NO.:189 and a heavy chainvariable region as set forth in SEQ ID NO.:196; j. a light chainvariable region as set forth in SEQ ID NO.:189 and a heavy chainvariable region as set forth in SEQ ID NO.:197; k. a light chainvariable region as set forth in SEQ ID NO.:190 and a heavy chainvariable region as set forth in SEQ ID NO.:194; l. a light chainvariable region as set forth in SEQ ID NO.:190 and a heavy chainvariable region as set forth in SEQ ID NO.:195; m. a light chain as setforth in SEQ ID NO.: 199 or SEQ ID NO.:200 and a heavy chain as setforth in SEQ ID NO.:202, SEQ ID NO.:203, SEQ ID NO.:204 or SEQ IDNO.:205; n. a light chain as set forth in SEQ ID NO.:199 and a heavychain as set forth in SEQ ID NO.:202; o. a light chain as set forth inSEQ ID NO.:199 and a heavy chain as set forth in SEQ ID NO.:203; p. alight chain as set forth in SEQ ID NO.:199 and a heavy chain as setforth in SEQ ID NO.:204; q. a light chain as set forth in SEQ ID NO.:199and a heavy chain as set forth in SEQ ID NO.:205; r. a light chain asset forth in SEQ ID NO.:200 and a heavy chain as set forth in SEQ IDNO.:202; s. a light chain as set forth in SEQ ID NO.:200 and a heavychain as set forth in SEQ ID NO.:203; t. a light chain as set forth inSEQ ID NO.:200 and a heavy chain as set forth in SEQ ID NO.:204 or; u. alight chain as set forth in SEQ ID NO.:200 and a heavy chain as setforth in SEQ ID NO.:205. 12-31. (canceled)
 32. The method of claim 11,wherein the antibody or antigen binding fragment thereof is conjugatedwith a therapeutic moiety.
 33. The method of claim 32, wherein thetherapeutic moiety is a cytotoxic agent.
 34. The method of claim 6,wherein the antibody or antigen binding fragment thereof has a highaffinity for KAAG1. 35-39. (canceled)
 40. The method of claim 1, whereinthe antibody or antigen binding fragment thereof is conjugated with atherapeutic moiety.
 41. The method of claim 40, wherein the antibody orantigen binding fragment thereof has a high affinity for KAAG1.
 42. Themethod of claim 1, wherein the antibody or antigen binding fragmentthereof comprises: a. a CDRH1 as set forth in SEQ ID NO.:49, a CDRH2 asset forth in SEQ ID NO.:50 or in SEQ ID NO.:212, a CDRH3 as set forth inSEQ ID NO.:51, a CDRL1 as set forth in SEQ ID NO.: 52, a CDRL2 as setforth in SEQ ID NO.:53 and a CDRL3 as set forth in SEQ ID NO.: 54; b. alight chain variable region as set forth in SEQ ID NO.:48 and a heavychain variable region as set forth in SEQ ID NO.:46; c. a light chainvariable region as set forth in SEQ ID NO.:186 wherein at least one ofthe amino acid identified by X is an amino acid substitution incomparison with a corresponding amino acid in the polypeptide set forthin SEQ ID NO.:48 and a heavy chain variable region as set forth in SEQID NO.:191 wherein at least one of the amino acid identified by X is anamino acid substitution in comparison with a corresponding amino acid inthe polypeptide set forth in SEQ ID NO.:46; d. a light chain variableregion as set forth in SEQ ID NO.:187 and a heavy chain variable regionas set forth in SEQ ID NO.:192; e. a light chain variable region as setforth in SEQ ID NO.:188 and a heavy chain variable region as set forthin SEQ ID NO.:193; f. a light chain variable region as set forth in SEQID NO.: 189 or SEQ ID NO.:190 and a heavy chain variable region as setforth in SEQ ID NO.:194, SEQ ID NO.:195, SEQ ID NO.:196 or SEQ IDNO.:197; g. a light chain variable region as set forth in SEQ ID NO.:189and a heavy chain variable region as set forth in SEQ ID NO.:194; h. alight chain variable region as set forth in SEQ ID NO.:189 and a heavychain variable region as set forth in SEQ ID NO.:195; i. a light chainvariable region as set forth in SEQ ID NO.:189 and a heavy chainvariable region as set forth in SEQ ID NO.:196; j. a light chainvariable region as set forth in SEQ ID NO.:189 and a heavy chainvariable region as set forth in SEQ ID NO.:197; k. a light chainvariable region as set forth in SEQ ID NO.:190 and a heavy chainvariable region as set forth in SEQ ID NO.:194; l. a light chainvariable region as set forth in SEQ ID NO.:190 and a heavy chainvariable region as set forth in SEQ ID NO.:195; m. a light chain as setforth in SEQ ID NO.: 199 or SEQ ID NO.:200 and a heavy chain as setforth in SEQ ID NO.:202, SEQ ID NO.:203, SEQ ID NO.:204 or SEQ IDNO.:205; n. a light chain as set forth in SEQ ID NO.:199 and a heavychain as set forth in SEQ ID NO.:202; o. a light chain as set forth inSEQ ID NO.:199 and a heavy chain as set forth in SEQ ID NO.:203; p. alight chain as set forth in SEQ ID NO.:199 and a heavy chain as setforth in SEQ ID NO.:204; q. a light chain as set forth in SEQ ID NO.:199and a heavy chain as set forth in SEQ ID NO.:205; r. a light chain asset forth in SEQ ID NO.:200 and a heavy chain as set forth in SEQ IDNO.:202; s. a light chain as set forth in SEQ ID NO.:200 and a heavychain as set forth in SEQ ID NO.:203; t. a light chain as set forth inSEQ ID NO.:200 and a heavy chain as set forth in SEQ ID NO.:204 or; u. alight chain as set forth in SEQ ID NO.:200 and a heavy chain as setforth in SEQ ID NO.:205.
 43. The method of claim 42, wherein theantibody or antigen binding fragment thereof is conjugated with atherapeutic moiety.
 44. A method of treating basal-like breast cancer,the method comprising administering an antibody or an antigen bindingfragment thereof capable of specific binding to Kidney associatedantigen 1 (KAAG1) to an individual in need.
 45. The method of claim 44,wherein the antibody or antigen binding fragment thereof is conjugatedwith a therapeutic moiety.
 46. The method of claim 45, wherein theantibody or antigen binding fragment thereof has a high affinity forKAAG1.
 47. The method of claim 44, wherein the antibody or antigenbinding fragment thereof comprises: a. a CDRH1 as set forth in SEQ IDNO.:49, a CDRH2 as set forth in SEQ ID NO.:50 or in SEQ ID NO.:212, aCDRH3 as set forth in SEQ ID NO.:51, a CDRL1 as set forth in SEQ ID NO.:52, a CDRL2 as set forth in SEQ ID NO.:53 and a CDRL3 as set forth inSEQ ID NO.: 54; b. a light chain variable region as set forth in SEQ IDNO.:48 and a heavy chain variable region as set forth in SEQ ID NO.:46;c. a light chain variable region as set forth in SEQ ID NO.:186 whereinat least one of the amino acid identified by X is an amino acidsubstitution in comparison with a corresponding amino acid in thepolypeptide set forth in SEQ ID NO.:48 and a heavy chain variable regionas set forth in SEQ ID NO.:191 wherein at least one of the amino acididentified by X is an amino acid substitution in comparison with acorresponding amino acid in the polypeptide set forth in SEQ ID NO.:46;d. a light chain variable region as set forth in SEQ ID NO.:187 and aheavy chain variable region as set forth in SEQ ID NO.:192; e. a lightchain variable region as set forth in SEQ ID NO.:188 and a heavy chainvariable region as set forth in SEQ ID NO.:193; f. a light chainvariable region as set forth in SEQ ID NO.: 189 or SEQ ID NO.:190 and aheavy chain variable region as set forth in SEQ ID NO.:194, SEQ IDNO.:195, SEQ ID NO.:196 or SEQ ID NO.:197; g. a light chain variableregion as set forth in SEQ ID NO.:189 and a heavy chain variable regionas set forth in SEQ ID NO.:194; h. a light chain variable region as setforth in SEQ ID NO.:189 and a heavy chain variable region as set forthin SEQ ID NO.:195; i. a light chain variable region as set forth in SEQID NO.:189 and a heavy chain variable region as set forth in SEQ IDNO.:196; j. a light chain variable region as set forth in SEQ ID NO.:189and a heavy chain variable region as set forth in SEQ ID NO.:197; k. alight chain variable region as set forth in SEQ ID NO.:190 and a heavychain variable region as set forth in SEQ ID NO.:194; l. a light chainvariable region as set forth in SEQ ID NO.:190 and a heavy chainvariable region as set forth in SEQ ID NO.:195; m. a light chain as setforth in SEQ ID NO.: 199 or SEQ ID NO.:200 and a heavy chain as setforth in SEQ ID NO.:202, SEQ ID NO.:203, SEQ ID NO.:204 or SEQ IDNO.:205; n. a light chain as set forth in SEQ ID NO.:199 and a heavychain as set forth in SEQ ID NO.:202; o. a light chain as set forth inSEQ ID NO.:199 and a heavy chain as set forth in SEQ ID NO.:203; p. alight chain as set forth in SEQ ID NO.:199 and a heavy chain as setforth in SEQ ID NO.:204; q. a light chain as set forth in SEQ ID NO.:199and a heavy chain as set forth in SEQ ID NO.:205; r. a light chain asset forth in SEQ ID NO.:200 and a heavy chain as set forth in SEQ IDNO.:202; s. a light chain as set forth in SEQ ID NO.:200 and a heavychain as set forth in SEQ ID NO.:203; t. a light chain as set forth inSEQ ID NO.:200 and a heavy chain as set forth in SEQ ID NO.:204 or; u. alight chain as set forth in SEQ ID NO.:200 and a heavy chain as setforth in SEQ ID NO.:205.
 48. The method of claim 47, wherein theantibody or antigen binding fragment thereof is conjugated with atherapeutic moiety.